Note: Descriptions are shown in the official language in which they were submitted.
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TITLE
REDUCED-MASS, LONG-ACTING DOSAGE FORMS
[0001] This application claims the benefit of and priority to U.S. Provisional
Application No. 60/933,647, filed June 7, 2007, which is hereby incorporated
herein by
reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to a method of delivering an antibody or
a nucleic
acid by administration of a free antibody or a free nucleic acid and a long-
acting (or
sustained-release) pharmaceutical dosage form of the antibody or nucleic acid.
BACKGROUND
[0003] The design and development of long-acting or sustained-release delivery
formulations have been the focus of considerable efforts in the pharmaceutical
industry for
decades. Parenteral formulations and, in particular, those that can be
administered by
injection or implantation, are particularly useful to achieve systemic and
local delivery of
bioactive agents for extended periods of times. The benefits of such dosage
forms are
multifold. Less frequent dosing afforded by long-acting formulations can
benefit the patient
by simply reducing the number and frequency of times that they need to be
administered
with the formulation. When administration involves injections or other
clinical procedures,
then, reducing frequency of injections is a benefit to the patient in terms of
reducing patient
discomfort, pain, and inconvenience - particularly for injections into the eye
or the spinal
cord or other sensitive sites. Moreover, the constant delivery of the
bioactive agent for long
periods of time can also improve compliance to the treatment program and,
consequently,
can improve recovery or treatment response.
[0004] Despite the long duration of certain sustained-release formulations of
bioactive
agents (e.g., up to 3, 6, or 9 months or longer), certain administrations are
problematic in
that the total volume of administration must be small. That is, the total
delivery volume for
certain administration routes is limited, and so, the amount of bioactive
agent that is
delivered is reduced due to the volume taken up by the polymer wall forming or
matrix
material and/or excipient of the microparticle or implant. The volume taken up
by the
microparticle or implant can lead to not enough bioactive agent being
delivered and/or the
bioactive agent not being delivered over a long enough period of time.
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[0005] There is a need to address the aforementioned problems and other
shortcomings
associated with the traditional delivery systems and the traditional methods
of delivering
certain bioactive agents. These needs and other needs are satisfied by the
delivery systems
and methods of the present invention.
SUMMARY OF THE INVENTION
[0006] The present invention relates to methods and compositions whereby
unencapsulated ("free") antibody or nucleic acid is co-administered with
encapsulated (such
as by microparticle or implant) antibody or nucleic acid to achieve a long
duration of
bioactive release. One result is that less encapsulation excipient is needed
allowing for
small-volume administrations as required, for example, for ocular, intra-
dermal, orthopedic,
brain, and spinal delivery. In one aspect, the unencapsulated antibody or
nucleic acid alone
has efficacy for an extended period, during which time, very little or no
encapsulated
bioactive is released. In one aspect, after the unencapsulated antibody or
nucleic acid has
reduced its activity, is gone, or no longer has activity, the encapsulated
antibody or nucleic
acid begins to release for a desired preprogrammed long acting duration. In
summary, less
formulation mass is needed because not all of the antibody or nucleic acid is
encapsulated
with encapsulation excipient. In addition, more antibody or nucleic acid can
be
administered to afford longer acting formulations. Lastly, the use of the
invention can be for
systemic or local delivery.
[0007] In one aspect, it is desirable to take advantage of the long half-life
of certain
antibodies or nucleic acids when designing and developing the long-acting
formulation. In
particular, it is beneficial to administer a free antibody or nucleic acid
that has a long
duration of action (either in the body or at a local site) at the same time as
administering or
delivering a long-acting formulation containing that same antibody or nucleic
acid. In
particular, because duration of action of a freely-administered antibody or
nucleic acid can
persist for some length of time, it is not necessary for the long-acting
formulation to be
designed to release the antibody or nucleic acid during this initial period of
time of free
antibody or nucleic acid duration.
[0008] In one aspect, the present invention describes a method for
administering free
antibody or nucleic acid concomitantly with a delayed-release formulation
comprising the
antibody or nucleic acid. Preferably, the long-acting formulation releases
relatively little of
the antibody or nucleic acid after administration so that the predominant
source of antibody
or nucleic acid that is released after administration is from the portion of
the antibody or
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nucleic acid that was administered as the free agent. In another aspect, the
invention
describes a controlled release formulation to achieve such an administration.
[0009] In one broad aspect, the aspect is directed to a method of extending
the release
profile of an antibody or nucleic acid in a subject while reducing the total
system mass of
the polymer material of a biodegradable, long-acting formulation comprising
administering
to the subject at about the same time a free antibody or nucleic acid and a
biodegradable,
long-acting formulation containing the antibody or nucleic acid, wherein the
free antibody
or nucleic acid has a pharmaceutically acceptable bioactivity period of at
least a week and
wherein the biodegradable, long-acting formulation releases its antibody or
nucleic acid to
coincide with the diminution of activity of the free antibody or nucleic acid.
[00010] In another broad aspect, the aspect is directed to a controlled
release
formulation comprising a free antibody or nucleic acid and a biodegradable,
long-acting
formulation containing the antibody or nucleic acid, wherein the free antibody
or nucleic
acid has a pharmaceutically acceptable bioactivity period of at least a week
and wherein the
biodegradable, long acting formulation releases its antibody or nucleic acid
to coincide with
the diminution of activity of the free antibody or nucleic acid
[00011] In another broad aspect, the aspect is directed to a method of
extending the
release profile of an antigen or nucleic acid while reducing the total system
mass of the
polymer wall forming material of a microparticle comprising administering at
about the
same time a free antigen or nucleic acid and a microparticle containing the
antigen or
nucleic acid, wherein the free antigen or nucleic acid has a pharmaceutically
acceptable
bioactivity period of at least a week and wherein the microparticle releases
its antigen or
nucleic acid to coincide with the diminution of activity of the free antigen
or nucleic acid.
[00012] In another broad aspect, the aspect is directed to a controlled
release
formulation comprising a free antigen or nucleic acid and a microparticle
containing the
antigen or nucleic acid, wherein the free antigen or nucleic acid has a
pharmaceutically
acceptable bioactivity period of at least a week and wherein the microparticle
releases its
antigen or nucleic acid to coincide with the diminution of activity of the
free antigen or
nucleic acid.
[00013] Otherwise, the advantages of the invention will be set forth in part
in the
description which follows, and in part will be obvious from the description,
or may be
learned by practice of the aspects described below. The advantages described
below will be
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realized and attained by means of the elements and combinations particularly
pointed out in
the appended claims. It is to be understood that both the foregoing general
description and
the following detailed description are exemplary and explanatory only and are
not
restrictive.
DETAILED DESCRIPTION OF THE INVENTION
[00014] The present invention can be understood more readily by reference to
the
following detailed description, examples, and claims, and their previous and
following
description. However, before the present compositions, articles, devices,
and/or methods
are disclosed and described, it is to be understood that this invention is not
limited to the
specific compositions, articles, devices, and/or methods disclosed unless
otherwise
specified, as such can, of course, vary. It is also to be understood that the
terminology used
herein is for the purpose of describing particular aspects only and is not
intended to be
limiting.
[00015] The following description of the invention is provided as an enabling
teaching
of the invention in its currently known embodiments. To this end, those
skilled in the
relevant art will recognize and appreciate that many changes can be made to
the various
aspects of the invention described herein, while still obtaining the
beneficial results of the
present invention. It will also be apparent that some of the desired benefits
of the present
invention can be obtained by selecting some of the features of the present
invention without
utilizing other features. Accordingly, those who work in the art will
recognize that many
modifications and adaptations to the present invention are possible and can
even be
desirable in certain circumstances and are a part of the present invention.
Thus, the
following description is provided as illustrative of the principles of the
present invention
and not in limitation thereof.
[00016] Definitions
[00017] Before the present compounds, compositions, and/or methods are
disclosed and
described, it is to be understood that the aspects described below are not
limited to specific
compounds, synthetic methods, or uses as such may, of course, vary. It is also
to be
understood that the terminology used herein is for the purpose of describing
particular
aspects only and is not intended to be limiting.
[00018] In this specification and in the claims that follow, reference will be
made to a
number of terms that shall be defined to have the following meanings:
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[00019] Throughout this specification, unless the context requires otherwise,
the word
"comprise," or variations such as "comprises" or "comprising," will be
understood to imply
the inclusion of a stated integer or step or group of integers or steps but
not the exclusion of
any other integer or step or group of integers or steps.
[00V0] It must be noted that, as used in the specification and the appended
claims, the
singular forms "a," "an" and "the" include plural referents unless the context
clearly dictates
otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes
mixtures of
two or more such carriers, and the like; reference to "an antibody or nucleic
acid" includes a
single antibody or nucleic acid or a mixture comprising two or more antibodies
or nucleic
acids and the like; similarly, "a polymer" includes a single polymer or a
mixture comprising
two or more polymers and the like; and so on.
[00021] "0ptional" or "optionally" means that the subsequently described event
or
circumstance can or cannot occur, and that the description includes instances
where the
event or circumstance occurs and instances where it does not.
[00022] Ranges may be expressed herein as from "about" one particular value,
and/or to
"about" another particular value. When such a range is expressed, another
aspect includes
from the one particular value and/or to the other particular value. Similarly,
when values
are expressed as approximations, by use of the antecedent "about," it will be
understood that
the particular value forms another aspect. It will be further understood that
the endpoints of
each of the ranges are significant both in relation to the other endpoint, and
independently
of the other endpoint.
[00023] As used herein, a "wt. %" or "weight percent" or "percent by weight"
of a
component, unless specifically stated to the contrary, refers to the ratio of
the weight of the
component to the total weight of the composition in which the component is
included,
expressed as a percentage.
[00024] By "contacting" is meant an instance of exposure by close physical
contact of
at least one substance to another substance.
[00025] By "sufficient amount" and "sufficient time" means an amount and time
needed to achieve the desired result or results, e.g., dissolve.a portion of
the polymer.
[00026] "Admixture" or "blend" is generally used herein to refer to a physical
combination of two or more different components. In the case of polymers, an
admixture,
or blend, of polymers is a physical blend or combination of two or more
different polymers.
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[00027] "Agent" is used herein to refer generally to compounds that are
contained in or
on the long-acting formulation. Agent may include an antibody or nucleic acid
or an
excipient or, more generally, any additive in the long-acting formulation.
"Agent" includes
a single such compound and is also intended to include a plurality of such
compounds.
[00028] "Bioactive agent" is used herein to include a compound of interest
contained in
or on a pharmaceutical formulation or dosage form that is used for
pharmaceutical or
medicinal purposes to provide some form of therapeutic effect or elicit some
type of
biologic response or activity. As used herein, bioactive agent typically
refers to an
antibody or nucleic acid. "Bioactive agent" includes a single such agent and
is also
intended to include a plurality of bioactive agents including, for example,
combinations of
two or more bioactive agents.
[00029] "Excipient" is used herein to include any other agent or compound that
may be
contained in a long-acting formulation that is not the antibody or nucleic
acid. As such, an.
excipient should be pharmaceutically or biologically acceptable or relevant
(for example, an
excipient should generally be non-toxic to the subject). "Excipient" includes
a single such
compound and is also intended to include a plurality of such compounds.
[00030] "Biocompatible" as used herein refers to a material that is generally
non-toxic
to the recipient and does not possess any significant untoward effects to the
subject and,
further, that any metabolites or degradation products of the material are non-
toxic to the
subject.
[00031] "Biodegradable" is generally referred to herein generally as a
material that will
erode to soluble species or that will degrade under physiologic conditions to
smaller units or
chemical species that are, themselves, non-toxic (biocompatible) to the
subject and capable
of being metabolized, eliminated, or excreted by the subject.
[00032] Terms such as "long-acting", "sustained-release" or "controlled
release" are
used generally to describe a formulation, dosage form, device or other type of
technologies
used, such as, for example, in the art to achieve the prolonged or extended
release or
bioavailability of an antibody or nucleic acid to a subject; it may refer to
technologies that
provide prolonged or extended release or bioavailability of an antibody or
nucleic acid to
the general systemic circulation or a subject or to local sites of action in a
subject including
(but not limited to) cells, tissues, organs, joints, regions, and the like.
Furthermore, these
terms may refer to a technology that is used to prolong or extend the release
of antibody or
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nucleic acid from a formulation or dosage form or they may refer to a
technology used to
extend or prolong the bioavailability or the pharmacokinetics or the duration
of action of a
antibody or nucleic acid to a subject or'they may refer to a technology that
is used to extend
or prolong the pharmacodynamic effect elicited by a formulation. A "long-
acting
formulation," a "sustained release fonmulation," or a "controlled release
formulation" (and
the like) is a pharmaceutical formulation, dosage form, or other technology
that is used to
provide long-acting release of an antibody or nucleic acid to a subject.
[00033] The term "modified bioactive agent" and the like is used herein to
refer,
generally, to a bioactive agent that has been modified with another entity
through either
covalent means or by non-covalent means. The term also is used to include
prodrug forms
of bioactive agents, where the prodrug form could be a polymeric prodrug or
non-polymeric
prodrug. Modifications conducted using polymers could be carried out with
synthetic
polymers (such as polyethylene glycol, PEG; polyvinylpyrrolidone, PVP;
polyethylene
oxide, PEO; propylene oxide, PPO; copolymers thereof; and the like) or
biopolymers (such
as polysaccharides, proteins, polypeptides, among others) or synthetic or
modified
biopolymers.
[00034] The tenm "microparticle" is used herein to refer generally to a
variety of
substantially structures having sizes from about 10 nm to 2000 microns (2
millimeters) and
includes microcapsule, microsphere, nanoparticle, nanocapsule, nanosphere as
well as
particles, in general, that are less than about 2000 microns (2 millimeters).
[00035] The terms "microencapsulated" and "encapsulated" are used herein to
refer
generally to an antibody or nucleic acid that is incorporated into any sort of
long-acting
formulation or technology regardless of shape or design; therefore,
a"microencapsulated"
or "encapsulated" antibody or nucleic acid may include antibody or nucleic
acid that is
incorporated into a particle or a microparticle and the like or it may include
antibody or
nucleic acid that is incorporated into a solid implant and so on.
[00036] "Implant" as used herein is intended to refer generally to a
controlled release
preformed macroscopic device.
[00037] "Needle" is used herein to refer to small-diameter devices that can be
used to
administer, deliver, inject, or otherwise introduce a long-acting formulation
to a subject
(either animal or human) for any purposes including medical, clinical,
surgical, therapeutic,
pharmaceutical, pharmacological, diagnostic, cosmetic, and prophylactic
purposes.
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Examples can include, without being limiting, needles, hypodermic needles,
surgical
needles, infusion needles, catheters, trocars, cannulas, tubes and tubing used
for clinical,
surgical, medical, procedural, or medical purposes, and the like.
[00038] "Subject" is used herein to refer to any target of administration. The
subject
can be a vertebrate, for example, a mammal. Thus, the subject can be a human.
The term
does not denote a particular age or sex. Thus, adult and newborn subjects, as
well as fetuses,
whether male or female, are intended to be covered. A "patient" refers to a
subject afflicted
with a disease or disorder and includes human and veterinary subjects.
[00039] Disclosed are compounds, compositions, and components that can be used
for,
can be used in conjunction with, can be used in preparation for, or are
products of the
disclosed methods and compositions. These and other materials are disclosed
herein, and it
is understood that when combinations, subsets, interactions, groups, etc. of
these materials
are disclosed that while specific reference of each various individual and
collective
combinations and permutation of these compounds may not be explicitly
disclosed, each is
specifically contemplated and described herein. For example, if a number of
different
polymers and agents are disclosed and discussed, each and every combination
and
permutation of the polymer and agent are specifically contemplated unless
specifically
indicated to the contrary. Thus, if a class of molecules A, B, and C are
disclosed as well as
a class of molecules D, E, and F and an example of a combination molecule, A-D
is
disclosed, then even if each is not individually recited, each is individually
and collectively
contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-
E, B-F,
C-D, C-E, and C-F are specifically contemplated and should be considered
disclosed from
disclosure of A, B, and C; D, E, and F; and the example combination A-D.
Likewise, any
subset or combination of these is also specifically contemplated and
disclosed. Thus, for
example, the sub-group of A-E, B-F, and C-E are specifically contemplated and
should be
considered disclosed from disclosure of A, B, and C; D, E, and F; and the
example
combination A-D. This concept applies to all aspects of this disclosure
including, but not
limited to, steps in methods of making and using the disclosed compositions.
Thus, if there
are a variety of additional steps that can be performed it is understood that
each of these
additional steps can be performed with any specific embodiment or combination
of
embodiments of the disclosed methods, and that each such combination is
specifically
contemplated and should be considered disclosed.
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[00040] Introduction and Discussiori
[00041] The present invention relates to a method of delivering an antibody or
nucleic
acid by administration of long-acting (or sustained-release) pharmaceutical
formulations.
More specifically, the present invention provides a method for administering
free antibody
or nucleic acid (unencapsulated bioactive) concomitantly with a long-acting
(delayed-
release followed by sustained release) formulation comprising the antibody or
nucleic acid.
In this manner, the bolus administration of the free antibody or nucleic acid
provides an
initial supply of antibody or nucleic acid to the subject, which persists for
a length of time
based on the pharmacokinetics (half-life and duration of action) of the free
antibody or
nucleic acid. In various aspects of the present invention, antibodies or
nucleic acids are
used that have a long duration of action from a bolus administration of the
free antibody or
nucleic acid lasting, for example, at least one week, at least two weeks, at
least three weeks,
at least four weeks, at least one month, or at least two months, or longer,
wherein the bolus
administration is able to provide pharmacokinetic antibody or nucleic acid
levels in the
bloodstream or in the local site that are sufficient to provide therapeutic
effects or an
otherwise desirable pharmacodynamic response or effect. In one aspect, such
long duration
antibodies or nucleic acids last at least a week and up to about a month, and
in certain cases
up to about three months.
[00042] After the duration of action of the bolus administration of an
antibody or nucleic
acid, the subsequent release of the antibody or nucleic acid from the long-
acting formulation
provides an additional supply of antibody or nucleic acid to the subject for
continued
therapeutic treatment. The duration of the long acting or sustained-release
formulation can
be any period of release capable in the art, for example, up to 3, 4, 5, 6, 7,
8, 9, 10, or 11
months or longer. In one aspect, the total release period can be made longer
with the
present invention because there is no need for any microparticle release
during the period of
or substantial period of free antibody or nucleic acid duration.
[00043] In one aspect, the long-acting formulation releases or delivers
relatively little of
the antibody or nucleic acid after it is first administered so that the
predominant source of
antibody or nucleic acid initially after administration is from the free
antibody or nucleic
acid that is administered in the bolus dose given concomitantly with the long-
acting
formulation. In this manner, the bolus administration of the free antibody or
nucleic acid
provides the initial antibody or nucleic acid to the body or the local site
and this portion of
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antibody or nucleic acid then persists for a length of time based on its
particular
pharmacokinetics profile (half-life and duration of action).
[00044] As used herein, the term "the biodegradable, long-acting formulation
releases its
antibody or nucleic acid to coincide with the diminution of activity of the
free antibody or
nucleic acid" includes aspects ranging from where the long-acting formulation
releases its
agent (1) prior to the free agent beginning to diminish its activity (in
anticipation of such a
free agent diminution in activity), (2) as the free agent begins to diminish
in activity, (3)
when the free agent is partially diminished in activity, for example at least
25%, at least
50% or at least 75% diminished, (4) when the free agent is substantially
diminished in
activity, or (5) when the free agent is completely gone or no longer has
activity. In one
aspect, the gap between the diminution of the free antibody or nucleic acid
and the
beginning of a sufficient release of the antibody or nucleic acid from the
long-acting
formulation is minimized. That is, in this aspect, it is undesirable for there
to be a period
between the free antibody or nucleic acid and long-acting formulation releases
of no or low
administration, such that, the antibody or nucleic acid is below
pharmaceutically acceptable
levels in the body. In a further aspect, there is at least an overlap or even
a substantial
overlap between the period of release of the free antibody or nucleic acid and
the period of
release from the long-acting formulation to ensure minimum acceptable levels
of the active
agent in the body are maintained. In another aspect, right after or shortly
after the
unencapsulated antibody or nucleic acid is gone or no longer has activity, the
encapsulated
antibody or nucleic acid begins to release for a desired preprogrammed long
acting duration.
[00045] In one aspect, the present invention describes a method in which the
concomitant administration of the bolus dose of free antibody or nucleic acid,
along with the
long-acting formulation is intended to provide for systemic delivery of the
antibody or
nucleic acid to the general circulation; or, instead, for local delivery to a
local site, tissue,
organ or the like; or for combinations thereof. In another regard, the present
invention
involves a long-acting formulation that is comprised of a non-degradable
biocompatible
polymer or a degradable biocompatible polymer or of combinations thereof. In
one regard,
the present invention involves the use of any type of long-acting dosage form
including, but
not limited to, particles (including microparticles, microspheres,
microcapsules,
nanoparticles, nanospheres, nanocapsules, and the like) or implants (including
injectable
implants and those that can be administered in surgical or clinical settings;
implants can
include solid, semisolid, hydrogel, viscous, liquid implants or combinations
thereof and
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may include implants that transition from one physical form to the other
before, during,.or
after administration). The long acting dosage form can be in the fon=n of an
injectable
liquid, gel, solution, or suspension.
[00046] Local delivery of an antibody or nucleic acid to locations such as
organs, cells
or tissues can also result in a therapeutically useful, long-lasting presence
of antibody or
nucleic acid, in those local sites or tissues since the routes by which an
antibody or nucleic
acid are distributed, metabolized, and eliminated from these locations may be
different than
the routes that define the pharmacokinetic duration of an antibody or nucleic
acid, delivered
to the general systemic circulation. The present invention can deliver to any
variety of sites,
locations, organs, cells, or tissues throughout the body. In one aspect, the
delivery is to
locations that historically are limited in the volume of administered
formulation, that is,
only a small amount of formulation volume is capable of being administered.
This aspect
includes, but is not limited to, a local delivery, an interarticular delivery,
such as between
the joints, orthopedic sites (bones, bone defects, joints, and the like), CNS
locations
(including, for example, spinal, cerebrospinal or intrathecal delivery or
delivery into the
brain or to specific sites in and around the brain), intradermal, intratumor,
peritumor, or
ocular delivery (to sites adjacent to or on the eye, sites within ocular
tissue, or intravitreal
delivery inside the eye).
[00047] In a specific aspect, the invention is directed to delivery to a
cancer or to the
vasculature associated with a cancer (e.g., to inhibit angiogenesis). The
cancer can be any
cell in a subject undergoing unregulated growth, invasion, or metastasis. In
some aspects,
the cancer can be any neoplasm or tumor for which radiotherapy is currently
used.
Alternatively, the cancer can be a neoplasm or tumor that is not sufficiently
sensitive to
radiotherapy using standard methods. Thus, the cancer can be a sarcoma,
lymphoma,
leukemia, carcinoma, blastoma, or germ cell tumor. In some aspects, the cancer
is a solid
tumor. In some aspects, the cancer is carcinoma. In some aspects, the cancer
is a sarcoma.
In some aspects, the cancer is a lymphoma. In some aspects, the cancer is germ-
cell tumor.
In some aspects, the cancer is blastic tumor. A representative but non-
limiting list of cancers
include B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin's
Disease,
myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head
and neck
cancer, squamous cell carcinoma of head and neck, kidney cancer, lung cancers
such as
small cell lung cancer and non-small cell lung cancer,
neuroblastoma/glioblastoma, ovarian
cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer,
melanoma, squamous
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cell carcinomas of the mouth, throat, larynx, and lung, colon cancer, cervical
carcinoma,
i
breast cancer, epithelial cancer, renal cancer, genitourinary cancer,
pulmonary cancer,
esophageal carcinoma, head and neck carcinoma, large bowel cancer,
hematopoietic
cancers; testicular cancer; colon and rectal cancers, prostatic cancer, and
pancreatic cancer.
[00048] In a specific aspect, the invention is directed to delivery to ocular
delivery.
Typically, free antibody or nucleic acid used to treat maladies of the eye
last only up to two
months and the total volume of administrated formulation is limited to up to
about 50 L or
up to about 100 L in certain cases. With the present invention, by using a
free antibody or
nucleic acid and a long-acting formulation, the volume limitations can still
be met while
producing a pharmaceutical effect of over two months. For example,
compositions of the
invention can be administered as a single injection into the eye to achieve
efficacy that lasts
for 3, 6, or 9 months or longer eliminating up to 3 injections into the eye.
[00049] With respect to the term, "administering at about the same time," as
used herein,
the method of the present invention may be practiced in a variety of ways
including, but not
limited to, delivering the free agent at the same time as the long-acting
formulation or
delivering the free agent sequentially to (either before or after) the
delivery of the long-
acting formulation. The time period between deliveries is intended to be
reasonably short.
Such administering at about the same time, includes, but is not limited to,
the following: the
combined administration of the free antibody or nucleic acid and the long-
acting
formulation in the same surgical or clinical procedure (for example, the co-
administration of
both in the same injection or during the same surgical intervention); or, in
separate
procedures that may be performed one after the other (as in the case of the
injection of the
bolus dose of the free antibody or nucleic acid in one procedure followed by
(or preceded
by) the administration of the long-acting formulation in a second procedure,
for example, by
the injection of a bolus dose of the free antibody or nucleic acid followed by
a second
injection of the long-acting formulation); or, concomitantly (at the same
time) but in
different procedures (as in the case of an infusion administration of the
bolus free antibody
or nucleic acid during which time the long-acting formulation is administered
by, for
example, injection or implantation). When the free antibody or nucleic acid
and the long-
acting formulation are performed in the same procedure, for example, in the
same injection,
the free antibody or nucleic acid can be combined with the long-acting
formulation in a
simple admixture. For example, the free antibody or nucleic acid can be
dissolved or
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suspended in a solvent such as water and the microencapsulated formulation can
be
suspended in the same water solution.
[00050] In one aspect, the administration is to a subject in need of such
administering.
[00051] Long-acting formulations
[00052] The method of the present invention includes the use of any type of
long-acting
formulation or dosage form that may be used (or envisioned to be used) for
delivery of a n
antibody or nucleic acid to prolong or extend an antibody or nucleic acid,
such as a antibody
or nucleic acid release, bioavailability, pharmacokinetics, pharmacodynamic
effects or
profiles.
[00053] Generally, long-acting or sustained release formulations comprise an
agent or
agents (including, for example, an antibody or nucleic acid) that is/are
incorporated or
associated with a biocompatible polymer in one manner or another. The matrix-
forming
polymers typically used in the preparation of long-acting formulations
include, but are not
limited, to biodegradable polymers (such as the polyesters poly(lactide),
poly(lactide-co-
glycolide), poly(caprolactone), poly(hydroxybutyrates), and the like) and non-
degradable
polymers (such as ethylenevinyl acetate (EVA), silicone polymers, and the
like). The agent
may be blended homogeneously throughout the polymer matrix or the agent may be
distributed unevenly (or discontinuously or heterogeneously) throughout the
polymer matrix
(as in the case of an antibody or nucleic acid-loaded core that is surrounded
by a polymer-
rich coating or polymer wall forming material as in the case of a
microcapsule, nanocapsule,
a coated or encapsulated implant, and the like). The dosage form may be in the
physical
form of particles, film, a fiber, a filament, a cylindrical implant, a
asymmetrically-shaped
implant, or a fibrous mesh (such as a woven or non-woven material; felt;
gauze, sponge,
and the like). When in the form of particles, the formulation may be in the
form of
microparticles, nanoparticles, microspheres, nanospheres, microcapsules or
nanocapsules,
and particles, in general, and combinations thereof. As such, the long-acting
(or sustained-
release) formulations of the present invention may include any variety of
types or designs
that are described, used or practiced in the art.
[00054] Long-acting formulations containing an antibody or nucleic acid can be
used to
deliver those agents to the systemic circulation or they can be used to
achieve local or site-
specific delivery to cells, tissues, organs, bones and the like that are
located nearby the site
of administration. Further, formulations can be used to achieve systemic
delivery of the
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antibody or nucleic acid and/or local delivery of the antibody or nucleic
acid. Formulations
can be delivered by injection (through, for example, needles, syringes,
trocars, cannula, and
the like) or by implantation. Delivery can be made via any variety of routes
of
administration commonly used for medical, clinical, surgical purposes
including, but not
limited to, intravenous, intraarterial, intramuscular, intraperitoneal,
subcutaneous,
intradermal, infusion and intracatheter delivery (and the like) in addition to
delivery to
specific locations (such as local delivery) including intrathecal,
intracardiac, intraosseous
(bone marrow), stereotactic-guided delivery, infusion delivery, CNS delivery,
stereo-
tactically administered delivery, orthopedic delivery (for example, delivery
to joints, into
bone, into bone defects and the like), cardiovascular delivery, inter- and
intra- and para-
ocular (including intravitreal and scleral and retrobulbar and sub-tenons
delivery and the
like), any delivery to any multitude of other sites, locations, organs,
tissues, etc.
[00055] In one aspect, the method of the present invention therefore envisions
utilizing
any technology that is used (or may be envisioned to be used) in the field for
parenteral
routes of administration including, for example but without being limited to
those described
by: Maindares and Silva, Curr Drug Targets, 5(5), 449 (2004); or, Degim and
Celebi, Curr
Pharm Des, 13(1), 99 (2007); or, Encyclopedia of Pharmaceutical Technology,
James
Swarbrick and James Boylan (Editors), Marcel Dekker, New York (2004); or,
Encyclopedia
of Controlled Drug Delivery, Edith Mathiowitz (Editor); John Wiley & Sons, New
York
(1999); or Controlled Release Veterinary Drug Delivery, Robert Gumy and
Michael J.
Rathbone (Editors); Elsevier Science B.V., Amsterdam, The Netherlands (2000);
or
Encyclopedia of Nanoscience and Nanotechnology, James Schwarz, Cristian
Contescu,
Karol Putyera (Editors), Marcel Dekker, Inc., New York (2004); or Encyclopedia
of
Biomaterials and Biomedical Engineering, Gary Wnek and Gary Bowlin (Editors),
Marcel
Dekker, Inc., New York (2004); or, Malik, Baboota, Ahuja, and Hassan, Curr
Drug Deliv.,
4(2), 141 (2007); or Nair and Laurencin, Adv Biochem Eng Biotechnol, 102, 47
(2006); and
the like. All of the above references are incorporated herein by this
reference for all of their
teachings as well as for the specific teachings of parenteral route technology
methods.
[00056] In one aspect, the method of the present invention includes long-
acting
formulations that can be administered by needle, injection, infusion,
implantation (as might
be conducted either clinically or surgically), and the like.
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[00057] Polymers and excipients
[00058] Polymers used to prepare the long-acting formulation can be any
biocompatible
polymer. One of skill in the art would know how to select without undue
experimentation
the proper polymer composition to achieve the desired effect of, in one
aspect, allowing the
free antibody or nucleic acid to provide its effect, and then, staging in the
release of the
antibody or nucleic acid from the long-acting formulation at an appropriate
time about on or
after the free antibody or nucleic acid provides its effect, as described
above. In one aspect
the polymer is selected to delay the release of the antibody or nucleic acid
until some time
after the free agent has provided its effect, thereby extending the total
effect period. Such
selection of the polymer can include criteria, such as, for example, the type
of polymer, the
selection of a polymer or a co-polymer, the type of co-monomers used in the co-
polymer,
the ratio of the types of monomers used in the co-polymer, the molecular
weight of the
polymer, the size of the microparticle, and any other criteria that is used by
one of skill in
the art to control the release profile of a microparticle.
[00059] Without intending to be limiting, examples may include any
biocompatible
polymers used in the art. For example, biocompatible non-degradable polymers
can be used
including, for example, a polyacrylate; a polymer of ethylene-vinyl acetate,
EVA; cellulose
acetate; an acyl-substituted cellulose acetate; a non-degradable polyurethane;
a polystyrene;
a polyvinyl chloride; a polyvinyl fluoride; a poly(vinyl imidazole); a
silicone-based polymer
(for example, Silastic and the like), a chlorosulphonate polyolefin; a
polyethylene oxide;
or a blend or copolymer thereof. Biocompatible biodegradable polymers can be
used
including, but not limited to, a poly(lactide); a poly(glycolide); a
poly(lactide-co-glycolide);
a poly(lactic acid); a poly(glycolic acid); a poly(lactic acid-co-glycolic
acid); a
poly(caprolactone); a poly(orthoester); a polyanhydride; a poly(phosphazene);
a
polyhydroxyalkanoate; a poly(hydroxybutyrate); a poly(hydroxybutyrate)
synthetically
derived; a poly(hydroxybutyrate) biologically derived; a polyester
synthetically derived; a
polyester biologically derived; a poly(lactide-co-caprolactone); a
poly(lactide-co-glycolide-
co-caprolactone); a polycarbonate; a tyrosine polycarbonate; a polyamide
(including
synthetic and natural polyamides, polypeptides, poly(amino acids) and the
like); a
polyesteramide; a polyester; a poly(dioxanone); a poly(alkylene alkylate); a
polyether (such
as polyethylene glycol, PEG, and polyethylene oxide, PEO); polyvinyl
pyrrolidone or PVP;
a polyurethane; a polyetherester; a polyacetal; a polycyanoacrylate; a
poly(oxyethylene)/poly(oxypropylene) copolymer; a polyacetal, a polyketal; a
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polyphosphate; a (phosphorous-containing) polymer; a polyphosphoester; a
polyhydroxyvalerate; a polyalkylene oxalate; a polyalkylene succinate; a
poly(maleic acid);
biopolymers or modified biopolymers including chitin, chitosan, modified
chitosan, among
other biocompatible polysaccharides; or biocompatible copolymers (including
block
copolymers or random copolymers) herein; or combinations or mixtures or
admixtures of
any polymers herein. Examples of copolymers that could be used include block
copolymers
containing blocks of hydrophilic or water-soluble polymers (such as
polyethylene glycol,
PEG, or polyvinyl pyrrolidone, PVP) with blocks of other biocompatible or
biodegradable
polymers (for example, poly(lactide) or poly(lactide-co-glycolide or
polycaprolcatone or
combinations thereof).
[00060] Furthermore, the present invention also relates to long-acting
formulations
prepared from copolymers that are comprised of the monomers of lactide
(including L-
lactide, D-lactide, and combinations thereof) or hydroxybutyrates or
caprolactone or
combinations thereof; and to long-acting formulations prepared from copolymers
that are
comprised of the monomers of DL-lactide, glycolide, hydroxybutyrate, and
caprolactone
and to long-acting formulations prepared from copolymers comprised of the
monomers of
DL-lactide or glycolide or caprolactone or hydroxybutyrates or combinations
therein.
Additionally, the present invention also relates to long-acting formulations
prepared from
admixtures containing the aforementioned copolymers (comprised of DL-lactide
or
glycolide or caprolactone or hydroxybutyrates or combinations therein) along
with other
biodegradable polymers including poly(DL-lactide-co-glycolide) or poly(DL-
lactide) or
PHA's, among others. The present invention can further include long-acting
formulations
prepared from block copolymers comprised with blocks of either hydrophobic or
hydrophilic biocompatible polymers or biopolymers or biodegradable polymers
such as
polyethers (including polyethylene glycol, PEG; polyethylene oxide, PEO;
polypropylene
oxide, PPO and block copolymers comprised of combinations thereof) or
polyvinyl
pyrrolidone (PVP), polysaccharides, conjugated polysaccharides, modified
polysaccharides,
such as fatty acid conjugated polysaccharides, polylactides, polyesters, among
others.
[00061] With the practice of the aspects herein, such as the combination of a
delivery of
the free antibody and nucleic acid along with the delivery of a long-acting
formulation of
the free antibody and nucleic acid, the polymer material (and in some aspects
the excipient
material) system mass is reduced due to less antibody or nucleic acid needed
in the long-
acting formulation.
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[00062] Free administration
[00063] The administration of the free antibody or nucleic acid (i.e.,
unencapsulated) is
performed using any method known in the art, such as by injection or infusion.
One of skill
in the art readily knows how to prepare a formulation of and administer a free
antibody or
nucleic acid. As discussed above, the free antibody or nucleic acid can be
delivered in the
same fonmulation as the long-acting formulation in one unitary formulation or
the free
antibody or nucleic acid can be delivered separately from the long-acting
formulation. In
one aspect, the free antibody or nucleic acid is delivered in the same
formulation as the
long-acting formulation as one unitary formulation.
[00064] The antibody or nucleic acid used in the free administration is
typically the
same or essentially the same as the antibody or nucleic acid used in the long-
acting
formulation. In one aspect the antibody or nucleic acid is the same as that
used in the long-
acting formulation.
[00065] Bioactive agents
[00066] Any antibody or nucleic acid for the free antibody or free nucleic
acid and the
long-acting formulation can be utilized. Some antibodies or nucleic acids have
long lasting
effects (days or weeks) in the body or in local tissues (when administered for
local delivery)
after administration, such as antibodies or nucleic acids that have half lives
ranging from
about 1.7 to 12 days or as long as 20 to 50 days or longer than 50 days.
Modified antibodies
or nucleic acids can also exhibit prolonged or extended pharmacokinetic
profiles, for
example, achieving a plasma half-life of about 3-6 days to as long as 14 days
or 1 month or
2 months or 3 months or longer. One aspect of the present invention involves
the use of an
antibody or nucleic acid that can provide a duration of action, i.e., a
pharmaceutically
acceptable bioactivity period (therapeutically efficacious blood or tissue
concentrations over
time) that extends for at least one week, at least two weeks, at least three
weeks, at least four
weeks, at least one month, at least two months, or at least three months or
longer following
a single bolus administration. In another aspect, any antibody or nucleic acid
can be used in
the present invention.
[00067] In one aspect, the antibody or nucleic acid is an antibody. In a
specific aspect,
the antibody is a therapeutic antibody. In another aspect, the antibody is a
therapeutic
antibody fragment. In another aspect, the antibody is a nanobody. In another
aspect, the
antibody is an Fab fragment. In another aspect, the antibody is an F(ab)2
fragment. In
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another aspect, the antibody is a single chain antibody. In another aspect,
the antibody is a
chimeric antibody. In another aspect, the antibody is a humanized antibody. In
another
aspect, the antibody is a recombinant antibody. In another aspect, the
antibody is a human
antibody. In another aspect, the antibody is a monoclonal antibody. In another
aspect, the
antibody is a polyclonal antibody.
[00068] In still another aspect, the antibody or nucleic acid is a nucleic
acid. In yet
another aspect, the nucleic acid is an aptamer, iRNA, siRNA, DNA, RNA,
antisense
nucleic acid or the like, or an antisense nucleic acid analog or the like. In
yet another
aspect, the nucleic acid is a small interfering RNA (siRNA). In yet another
aspect, the
nucleic acid is an antisense oligonucleotide. In yet another aspect, the
nucleic acid is a
nucleic acid encoding a protein. In yet another aspect, the nucleic acid is a
vector
comprising a nucleic acid encoding a protein operably linked to an expression
control
sequence.
[00069] In yet another aspect, the antibody or nucleic acid is a modified
antibody or
nucleic acid. In one aspect, the antibody is a PEG-modified antibody. In
another aspect,
the antibody is a PEG-modified antibody fragment. In still another aspect, the
nucleic acid
is a PEG-modified nucleic acid.
[00070] The antibody or nucleic acid is used for the treatment, diagnosis,
cure or
mitigation of disease or illness, a substance which affects the structure or
function of the
body, or pro-drugs, which become biologically active or more active after they
have been
placed in a predetermined physiological environment. In a specific aspect, the
antibody or
nucleic acid is not a vaccine. Antibodies or nucleic acids include
biologically,
physiologically, or pharmacologically active substances that act locally or
systemically in
the human or animal body. Various forms of the antibodies or nucleic acids can
be used,
which are capable of being released from the solid matrix into adjacent
tissues or fluids. A
liquid or solid antibody or nucleic acid can be incorporated in the delivery
systems
described herein. The antibodies or nucleic acids are at least very slightly
water soluble,
preferably moderately water soluble, and are diffusible through the polymeric
composition.
They can be acidic, basic, or amphoteric salts. They can be in the free acid
or free base
form. They can be nonionic molecules, polar molecules, or molecular complexes
capable of
hydrogen bonding. The antibody or nucleic acid may be included in the
compositions in the
form of, for example, an uncharged molecule, a molecular complex, a salt, an
ether, an
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ester, an amide, polymer-drug conjugate, or other form to prdvide the
effective biological or
physiological activity.
[00071] The long-acting formulations of the present invention can comprise one
antibody or nucleic acid or combinations of two or more antibodies or nucleic
acids
including a large number of antibodies or nucleic acids. The antibody or
nucleic acid can be
naturally-occurring, produced from fermentation or bacterial sources, or
synthetic in origin
or they can be prepared from a combination therein. The antibody or nucleic
acid can be a
compound that has been covalently or non-covalently modified using other
materials.
Examples include salt counter-ions, targeting agents, solubility modifiers,
permeability
modifiers, hydrophobic agents, hydrophilic agents, hydrophobic polymers,
hydrophilic
polymers, block copolymers, and the like.
[00072] The antibody of the disclosed compositions and methods can be an
antibody,
such as a therapeutic antibody. The term "antibody" is used herein in a broad
sense and
includes both polyclonal and monoclonal antibodies. In addition to intact
immunoglobulin
molecules, also included in the term "antibodies" are fragments or polymers of
those
immunoglobulin molecules, and human or humanized versions of immunoglobulin
molecules or fragments thereof, as long as they are chosen for their ability
to interact with
an antigen such that the antigen is inhibited from interacting with its
target, such as a ligand
or receptor. The antibodies can be tested for their desired activity using the
in vitro assays
described herein, or by analogous methods, after which their in vivo
therapeutic and/or
prophylactic activities are tested according to known clinical testing
methods.
[00073] As used herein, therapeutic antibodies are antibodies that are
administered to a
subject based on the ability of the antibody to bind a target antigen. They
are therefore
distinct from vaccines which are administered to a subject to induce an immune
response in
the subject thereby generating endogenous antibodies to the antigen.
Therapeutic antibodies
are known in the art and continue to be identified. The herein disclosed
methods can be used
with any antibody discovered to have a therapeutic effect when it binds its
antigen. The
antigen can be on a cell, such as a cancer cell. The antigen can be a growth
factor. The
antigen can be an extracellular structural protein.
[00074] The antigen of the disclosed antibody can be, for example, tumour
necrosis
factor alpha (TNFa). Infliximab (REMICADE) is a chimeric monoclonal antibody
(murine
binding VK and VH domains and human constant Fc domains) used to treat
autoimmune
disorders.. Infliximab binds TNFa thereby preventing it from signaling its
receptors on the
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surface of cells. TNFa is one of the key cytokines that triggers and sustains
the
inflammation response. Infliximab has been approved by the U.S. Food and Drug
Administration for the treatment of psoriasis, Crohn's disease, ankylosing
spondylitis,
psoriatic arthritis, rheumatoid arthritis, and ulcerative colitis.
[00075] The antigen of the disclosed antibody can be, for example, a vascular
endothelial growth factor (VEGF), such as VEGF-A. VEGF is involved in the
growth of
new blood vessels and vascular permeability. As such, inhibition of its
activity is useful for
treating or preventing leaky blood vessels or tumor angiogenesis.
[00076] In a specific aspect, the antibody is VEGF-trap (AFLIBERCEPT). VEGF-
trap is
a protein comprised of segments of the extracellular domains of human vascular
endothelial
growth factor receptors 1(VEGFRI) and 2 (VEGFR2) fused to the constant region
(Fc) of
human IgGl with potential antiangiogenic activity. Afilbercept, functioning as
a soluble
decoy receptor, binds to pro-angiogenic vascular endothelial growth factors
(VEGFs),
thereby preventing VEGFs from binding to their cell receptors. Disruption of
the binding of
VEGFs to their cell receptors may result in the inhibition of tumor
angiogenesis, metastasis,
and ultimately tumor regression.
[00077] Bevacizumab (trade name Avastin) is a monoclonal antibody against
VEGF. It
is used in the treatment of cancer, where it inhibits tumor growth by blocking
the formation
of new blood vessels. Bevacizumab can be used in combination with standard
chemotherapy in the treatment of metastatic colon cancer and most forms of
metastatic non-
small cell lung cancer. Bevacizumab can be used at least to treat in breast
cancer, metastatic
renal cell carcinoma, metastatic glioblastoma multiforme, metastatic ovarian
cancer,
metastatic hormone-refractory prostate cancer, and metastatic or unresectable
locally
advanced pancreatic cancer.
[00078] Ranibizumab (LUCENTIS) is a monoclonal antibody fragment derived from
the
same parent murine antibody as bevacizumab (AVASTIN). It is much smaller than
the
parent molecule and has been affinity matured to provide stronger binding to
VEGF-A. It
has been approved to treat the "wet" type of age-related macular degeneration
(ARMD), a
common form of age-related vision loss. Ranibizumab binds to and inhibits all
subtypes of
vascular endothelial growth factor A (VEGF-A). VEGF can trigger the growth of
new
vessels, which can leak blood and fluid into the eye. These leaky blood
vessels can
contribute to macular edema and choroidal neovascularization, resulting in the
wet type of
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ARMD. By blocking VEGF-A in the eye, ranibizumab can prevent and reverse
vision loss
caused by wet macular degeneration.
[00079] The antigen of the disclosed antibody can be, for example, cluster of
differentiation 20 (CD20). CD20 is widely expressed on B-cells. Rituximab
(RITUXAN,
MABTHERA) is a chimeric monoclonal antibody used in the treatment of B cell
non-
Hodgkin's lymphoma, B cell leukemia, and some autoimmune disorders. Rituximab
depletes B cells, and therefore is used to treat diseases which are
characterized by having
too many B cells, overactive B cells or dysfunctional B cells. Rituximab can
be used to treat
rheumatoid arthritis and autoimmune diseases, including idiopathic autoimmune
hemolytic
anemia, Pure red cell aplasia, idiopathic thrombocytopenic purpura (ITP),
Evans syndrome,
vasculitis, multiple sclerosis, bullous skin disorders (for example pemphigus,
pemphigoid),
type 1 diabetes mellitus, Sjogren's syndrome, Devic's Syndrome and systemic
lupus
erythematosus. Rituximab can also be used in the management of Renal
Transplant
recipients.
[00080] Ibritumomab tiuxetan (ZEVALIN) is a monoclonal antibody that binds to
the
CD20 antigen found on the surface of normal and malignant B cells (but not B
cell
precursors), allowing radiation from the attached isotope (mostly beta
emission) to kill it
and some nearby cells. This antibody can be used in radioimmunotherapy
treatment for
some forms of B cell non-Hodgkin's lymphoma. The drug uses the monoclonal
mouse IgGI
antibody ibritumomab in conjunction with the chelator tiuxetan, to which a
radioactive
isotope (either yttrium-90 or indium-111) is added. In addition, the antibody
itself can
trigger cell death via antibody-dependent cell-mediated cytotoxicity (ADCC),
complement-
dependent cytotoxicity (CDC), and apoptosis. Together, these actions eliminate
B cells from
the body, allowing a new population of healthy B cells to develop from
lymphoid stem
cells.
[00081] The antigen of the disclosed antibody can be, for example, beta-
amyloids. The
pathology of Alzheimer's disease shows a significant correlation between beta-
amyloid
peptide conformation and the clinical severity of dementia. Site-directed
antibodies can
modulate formation of beta-amyloid. Moreover, the antibodies can dissolve fl-
amyloid
plaques and protect the subject from learning and age-related memory deficits.
[00082] The antigen of the disclosed antibody can be, for example, a4-
integrin.
Natalizumab is a humanized monoclonal antibody against the cellular adhesion
molecule
a4-integrin. Natalizumab is used in the treatment of multiple sclerosis and
Crohn's disease.
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Natalizumab has prevents relapse, vision loss, cognitive decline and
significantly improving
quality of life in people with multiple sclerosis, as well as increasing rates
of remission and
preventing relapse in Crohn's disease.
[00083] The antigen of the disclosed antibody can be, for example, HER2/neu.
Trastuzumab (HERCEPTIN) is a humanized monoclonal antibody that acts on the
HER2/neu (erbB2) receptor. Trastuzumab's principal use is as an anti-cancer
therapy in
breast cancer in patients whose tumors over express (produce more than the
usual amount
of) this receptor. Amplification of HER2/neu (ErbB2) occurs in 20-30% of early-
stage
breast cancers. It encodes the extracellular domain of HER2.
[00084] The antigen of the disclosed antibody can be, for example, epidermal
growth
factor receptor (EGFR). Cetuximab (ERBITUX) is a chimeric monoclonal antibody
specific
for EGFR given by intravenous injection for treatment of metastatic colorectal
cancer and
head and neck cancer. Panitumumab (VECTIBIX) is another EGFR antibody being
used.
One of the main differences is that Cetuximab is an IgGI antibody, and
Panitumumab an
IgG2 antibody. Cetuximab is binds the extracellular domain of the EGFR of all
cells that
express EGFR, which includes the subset "cancer cells", preventing ligand
binding and
activation of the receptor. This blocks the downstream signaling of EGFR
resulting in
impaired cell growth and proliferation. Cetuximab has also been shown to
mediate antibody
dependent cellular cytotoxicity (ADCC).
[00085] The antigen of the disclosed antibody can be, for example, CD52.
Alemtuzumab
(CAMPATH, MABCAMPATH, or CAMPATH-1H) is a monoclonal antibody that targets
CD52, a protein present on the surface of mature lymphocytes, but not on the
stem cells
from which these lymphocytes were derived. It is used in the treatment of
chronic
lyinphocytic leukemia (CLL) and T-cell lymphoma. Alemtuzumab is also used in
some
conditioning regimens for bone marrow transplantation and kidney
transplantation. It can
also be used for treatment of autoimmune diseases, such as multiple sclerosis.
[00086] The term "monoclonal antibody" as used herein refers to an antibody
obtained
from a substantially homogeneous population of antibodies, i.e., the
individual antibodies
within the population are identical except for possible naturally occurring
mutations that
may be present in a small subset of the antibody molecules. The monoclonal
antibodies
herein specifically include "chimeric" antibodies in which a portion of the
heavy and/or
light chain is identical with or homologous to corresponding sequences in
antibodies
derived from a particular species or belonging to a particular antibody class
or subclass,
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while the remainder of the chain(s) is identical with or homologous to
corresponding
sequences in antibodies derived from another species or belonging to another
antibody class
or subclass, as well as fragments of such antibodies, as long as they exhibit
the desired
antagonistic activity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc.
Natl. Acad.
Sci. USA, 81:6851-6855 (1984)).
[00087] The disclosed monoclonal antibodies can be made using any procedure
which
produces monoclonal antibodies. For example, disclosed monoclonal antibodies
can be
prepared using hybridoma methods, such as those described by Kohler and
Milstein,
Nature, 256:495 (1975). In a hybridoma method, a mouse or other appropriate
host animal
is typically immunized with an immunizing agent to elicit lymphocytes that
produce or are
capable of producing antibodies that will specifically bind to the immunizing
agent.
Alternatively, the lymphocytes may be immunized in vitro.
[00088] If these approaches do not produce neutralizing antibodies, cells
expressing cell
surface localized versions of these proteins will be used to immunize mice,
rats or other
species. Traditionally, the generation of monoclonal antibodies has depended
on the
availability of purified protein or peptides for use as the immunogen. More
recently DNA
based immunizations have shown promise as a way to elicit strong immune
responses and
generate monoclonal antibodies. In this approach, DNA-based immunization can
be used,
wherein DNA encoding extracellular fragments of the antigen expressed as a
fusion protein
with human IgGI or an epitope tag is injected into the host animal according
to methods
known in the art (e.g., Kilpatrick KE, et al. Gene gun delivered DNA-based
immunizations
mediate rapid production of murine monoclonal antibodies to the Flt-3
receptor.
Hybridoma. 1998 Dec;17(6):569-76; Kilpatrick KE et al. High-affinity
monoclonal
antibodies to PED/PEA-15 generated using 5 microg of DNA. Hybridoma. 2000
Aug;19(4):297-302, which are incorporated herein by referenced in full for the
methods of
antibody production) and as described in the examples.
[00089] An alternate approach to immunizations with either purified protein or
DNA is
to use antigen expressed in baculovirus. The advantages to this system include
ease of
generation, high levels of expression, and post-translational modifications
that are highly
similar to those seen in mammalian systems. Use of this system involves
expressing the
extracellular domain of the antigen as fusion proteins with a signal sequence
fragment. The
antigen is produced by inserting a gene fragment in-frame between the signal
sequence and
the mature protein domain of the antigen's nucleotide sequence. This results
in the display
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of the foreign proteins on the surface of the virion. This method allows
immunization with
whole virus, eliminating the need for purification of target antigens.
[00090] Generally, either peripheral blood lymphocytes ("PBLs") are used in
methods
of producing monoclonal antibodies if cells of human origin are desired, or
spleen cells or
lymph node cells are used if non-human manunalian sources are desired. The
lymphocytes
are then fused with an immortalized cell line using a suitable fusing agent,
such as
polyethylene glycol, to form a hybridoma cell (Goding, "Monoclonal Antibodies:
Principles
and Practice" Academic Press, (1986) pp. 59-103). Immortalized cell lines are
usually
transformed mammalian cells, including myeloma cells of rodent, bovine,
equine, and
human origin. Usually, rat or mouse myeloma cell lines are employed. The
hybridoma cells
may be cultured in a suitable culture medium that preferably contains one or
more
substances that inhibit the growth or survival of the unfused, immortalized
cells. For
example, if the parental cells lack the enzyme hypoxanthine guanine
phosphoribosyl
transferase (HGPRT or HPRT), the culture medium for the hybridomas typically
will
include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which
substances
prevent the growth of HGPRT-deficient cells. Preferred immortalized cell lines
are those
that fuse efficiently, support stable high level expression of antibody by the
selected
antibody-producing cells, and are sensitive to a medium such as HAT medium.
More
preferred immortalized cell lines are murine myeloma lines, which can be
obtained, for
instance, from the Salk Institute Cell Distribution Center, San Diego, Calif
and the
American Type Culture Collection, Rockville, Md. Human myeloma and mouse-human
heteromyeloma cell lines also have been described for the production of human
monoclonal
antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., "Monoclonal
Antibody
Production Techniques and Applications" Marcel Dekker, Inc., New York, (1987)
pp. 51-
63). The culture medium in which the hybridoma cells are cultured can then be
assayed for
the presence of monoclonal antibodies directed against the antigen.
Preferably, the binding
specificity of monoclonal antibodies produced by the hybridoma cells is
determined by
immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay
(RIA) or
enzyme-linked inununoabsorbent assay (ELISA). Such techniques and assays are
known in
the art, and are described further in the Examples below or in Harlow and Lane
"Antibodies,
A Laboratory Manual" Cold Spring Harbor Publications, New York, (1988).
[00091] After the desired hybridoma cells are identified, the clones may be
subcloned by
limiting dilution or FACS sorting procedures and grown by standard methods.
Suitable
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culture media for this purpose include, for example, Dulbecco's Modified
Eagle's Medium
and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo
as
ascites in a mammal.
[00092] The monoclonal antibodies secreted by the subclones may be isolated or
purified from the culture medium or ascites fluid by conventional
immunoglobulin
purification procedures such as, for example, protein A-Sepharose, protein G,
hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity
chromatography.
[00093] The monoclonal antibodies may also be made by recombinant DNA methods,
such as those described in U.S. Pat. No. 4,816,567 (Cabilly et al.). DNA
encoding the
disclosed monoclonal antibodies can be readily isolated and sequenced using
conventional
procedures (e.g., by using oligonucleotide probes that are capable of binding
specifically to
genes encoding the heavy and light chains of murine antibodies). Libraries of
antibodies or
active antibody fragments can also be generated and screened using phage
display
techniques, e.g., as described in U.S. Patent No. 5,804,440 to Burton et al.
and U.S. Patent
No. 6,096,441 to Barbas et al.
[00094] In vitro methods are also suitable for preparing monovalent
antibodies.
Digestion of antibodies to produce fragments thereof, particularly, Fab or
F(ab)2 fragments,
can be accomplished using routine techniques known in the art. For instance,
digestion can
be performed using papain. Examples of papain digestion are described in WO
94/29348
published Dec. 22, 1994 and U.S. Pat. No. 4,342,566. Papain digestion of
antibodies
typically produces two identical antigen binding fragments, called Fab
fragments, each with
a single antigen binding site, and a residual Fc fragment. Pepsin treatment
yields an Fc
fragment and an F(ab)2 fragment that has two antigen combining sites and is
still capable of
cross-linking antigen.
[00095] The fragments, whether attached to other sequences or not, can also
include
insertions, deletions, substitutions, or other selected modifications of
particular regions or
specific amino acids residues, provided the activity of the antibody or
antibody fragment is
not significantly altered or impaired compared to the non-modified antibody or
antibody
fragment. These modifications can provide for some additional property, such
as to
remove/add amino acids capable of disulfide bonding, to increase its bio-
longevity, to alter
its secretory characteristics, etc. In any case, the antibody or antibody
fragment must
possess a bioactive property, such as specific binding to its cognate antigen.
Functional or
active regions of the antibody or antibody fragment may be identified by
mutagenesis of a
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specific region of the protein, followed by expression and testing of the
expressed
polypeptide. Such methods are readily apparent to a skilled practitioner in
the art and can
include site-specific mutagenesis of the nucleic acid encoding the antibody or
antibody
fragment. (Zoller, M.J. Curr. Opin. Biotechnol. 3:348-354, 1992).
[00096] As used herein, the term "antibody" or "antibodies" can also refer to
a human
antibody and/or a humanized antibody. Many non-human antibodies (e.g., those
derived
from mice, rats, or rabbits) are naturally antigenic in humans, and thus can
give rise to
undesirable immune responses when administered to humans. Therefore, the use
of human
or humanized antibodies in the methods serves to lessen the chance that an
antibody
administered to a human will evoke an undesirable immune response.
i. Whole Immuno log bulin
[00097] As used herein, the tenn "antibody" encompasses, but is not limited
to, whole
immunoglobulin (i.e., an intact antibody) of any class. Native antibodies are
usually
heterotetrameric glycoproteins, composed of two identical light (L) chains and
two identical
heavy (H) chains. Typically, each light chain is linked to a heavy chain by
one covalent
disulfide bond, while the number of disulfide linkages varies between the
heavy chains of
different immunoglobulin isotypes. Each heavy and light chain also has
regularly spaced
intrachain disulfide bridges. Each heavy chain has at one end a variable
domain (V(H))
followed by a number of constant domains. Each light chain has a variable
domain at one
end (V(L)) and a constant domain at its other end; the constant domain of the
light chain is
aligned with the first constant domain of the heavy chain, and the light chain
variable
domain is aligned with the variable domain of the heavy chain. Particular
amino acid
residues are believed to form an interface between the light and heavy chain
variable
domains. The light chains of antibodies from any vertebrate species can be
assigned to one
of two clearly distinct types, called kappa (k) and lambda (1), based on the
amino acid
sequences of their constant domains. Depending on the amino acid sequence of
the constant
domain of their heavy chains, immunoglobulins can be assigned to different
classes. There
are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM,
and several
of these may be further divided into subclasses (isotypes), e.g., IgG-1, IgG-
2, IgG-3, and
IgG-4; IgA-1 and IgA-2. One skilled in the art would recognize the comparable
classes for
mouse. The heavy chain constant domains that correspond to the different
classes of
immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively.
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[00098] The term "variable" is used herein to describe certain portions of the
variable
domains that differ in sequence among antibodies and are used in the binding
and
specificity of each particular antibody for its particular antigen. However,
the variability is
not usually evenly distributed through the variable domains of antibodies. It
is typically
concentrated in three segments called complementarity determining regions
(CDRs) or
hypervariable regions both in the light chain and the heavy chain variable
domains. The
more highly conserved portions of the variable domains are called the
framework (FR). The
variable domains of native heavy and light chains each comprise four FR
regions, largely
adopting a b-sheet configuration, connected by three CDRs, which form loops
connecting,
and in some cases forming part of, the b-sheet structure. The CDRs in each
chain are held
together in close proximity by the FR regions and, with the CDRs from the
other chain,
contribute to the formation of the antigen binding site of antibodies (see
Kabat E. A. et al.,
"Sequences of Proteins of Immunological Interest," National Institutes of
Health, Bethesda,
Md. (1987)). The constant domains are not involved directly in binding an
antibody to an
antigen, but exhibit various effect or functions, such as participation of the
antibody in
antibody-dependent cellular toxicity.
ii. Antibody Fragments
[00099] The term "antibody" as used herein is meant to include intact
molecules as well
as fragments thereof, such as, for example, Fab and F(ab')2, which are capable
of binding
the epitopic determinant.
[000100] As used herein, the term "antibody or fragments thereof' encompasses
chimeric
antibodies and hybrid antibodies, with dual or multiple antigen or epitope
specificities, and
fragments, such as F(ab')2, Fab', Fab and the like, including hybrid
fragments. Thus,
fragments of the antibodies that retain the ability to bind their specific
antigens are
provided. For example, fragments of antibodies which maintain antigen binding
activity are
included within the meaning of the term "antibody or fragment thereof." Such
antibodies
and fragments can be made by techniques known in the art and can be screened
for
specificity and activity according to the methods set forth in the Examples
and in general
methods for producing antibodies and screening antibodies for specificity and
activity (See
Harlow and Lane. Antibodies, A Laboratory Manual. Cold Spring Harbor
Publications,
New York, (1988)).
[000101] Also included within the meaning of "antibody or fragments thereof'
are
conjugates of antibody fragments and antigen binding proteins (single chain
antibodies) as
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described, for example, in U.S. Pat. No. 4,704,692, the contents of which are
hereby
incorporated by reference.
[000102] An isolated immunogenically specific paratope or fragment of the
antibody is
also provided. A specific immunogenic epitope of the antibody can be isolated
from the
whole antibody by chemical or mechanical disruption of the molecule. The
purified
fragments thus obtained are tested to determine their immunogenicity and
specificity by the
methods taught herein. Immunoreactive paratopes of the antibody, optionally,
are
synthesized directly. An immunoreactive fragment is defined as an amino acid
sequence of
at least about two to five consecutive amino acids derived from the antibody
amino acid
sequence.
[000103] Alternatively, unprotected peptide segments are chemically linked
where the
bond formed between the peptide segments as a result of the chemical ligation
is an
unnatural (non-peptide) bond (Schnolzer, M et al. Science, 256:221 (1992)).
This technique
has been used to synthesize analogs of protein domains as well as large
amounts of
relatively pure proteins with full biological activity (deLisle Milton RC et
al., Techniques in
Protein Chemistry IV. Academic Press, New York, pp. 257-267 (1992)).
[000104] Also disclosed are fragments of antibodies which have bioactivity.
The
polypeptide fragments can be recombinant proteins obtained by cloning nucleic
acids
encoding the polypeptide in an expression system capable of producing the
polypeptide
fragments thereof, such as an adenovirus or baculovirus expression system. For
example,
one can determine the active domain of an antibody from a specific hybridoma
that can
cause a biological effect associated with the interaction of the antibody with
antigen. For
example, amino acids found to not contribute to either the activity or the
binding specificity
or affinity of the antibody can be deleted without a loss in the respective
activity. For
example, in various embodiments, amino or carboxy-terminal amino acids are
sequentially
removed from either the native or the modified non-immunoglobulin molecule or
the
immunoglobulin molecule and the respective activity assayed in one of many
available
assays. In another example, a fragment of an antibody comprises a modified
antibody
wherein at least one amino acid has been substituted for the naturally
occurring amino acid
at a specific position, and a portion of either amino terminal or carboxy
terminal amino
acids, or even an internal region of the antibody, has been replaced with a
polypeptide
fragment or other moiety, such as biotin, which can facilitate in the
purification of the
modified antibody. For example, a modified antibody can be fused to a maltose
binding
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protein, through either peptide chemistry or cloning the respective nucleic
acids encoding
the two polypeptide fragments into an expression vector such that the
expression of the
coding region results in a hybrid polypeptide. The hybrid polypeptide can be
affinity
purified by passing it over an amylose affinity column, and the modified
antibody receptor
can then be separated from the maltose binding region by cleaving the hybrid
polypeptide
with the specific protease factor Xa. (See, for example, New England Biolabs
Product
Catalog, 1996, pg. 164.). Similar purification procedures are available for
isolating hybrid
proteins from eukaryotic cells as well.
[000105] The fragments, whether attached to other sequences or not, include
insertions,
deletions, substitutions, or other selected modifications of particular
regions or specific
amino acids residues, provided the activity of the fragment is not
significantly altered or
impaired compared to the nonmodified antibody or antibody fragment. These
modifications
can provide for some additional property, such as to remove or add amino acids
capable of
disulfide bonding, to increase its bio-longevity, to alter its secretory
characteristics, etc. In
any case, the fragment must possess a bioactive property, such as binding
activity,
regulation of binding at the binding domain, etc. Functional or active regions
of the
antibody may be identified by mutagenesis of a specific region of the protein,
followed by
expression and testing of the expressed polypeptide. Such methods are readily
apparent to a
skilled practitioner in the art and can include site-specific mutagenesis of
the nucleic acid
encoding the antigen. (Zoller MJ et al. Nucl. Acids Res. 10:6487-500 (1982).
[000106] Techniques can also be adapted for the production of single-chain
antibodies
specific to an antigenic protein of the present disclosure (see e.g., U. S.
Pat. No. 4,946,778).
In addition, methods can be adapted for the construction of F (ab) expression
libraries (see
e.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid and effective
identification
of monoclonal F (ab )fragments with the desired specificity for a protein or
derivatives,
fragments, analogs or homologs thereof. Antibody fragments that contain the
idiotypes to a
protein antigen may be produced by techniques known in the art including, but
not limited
to: (i) an F ((ab'))(2 )fragment produced by pepsin digestion of an antibody
molecule; (ii) an
Fab fragment generated by reducing the disulfide bridges of an F ((ab'))(2
)fragment; (iii) an
F (ab )fragment generated by the treatment of the antibody molecule with
papain and a
reducing agent and (iv) F (v), fragments.
[000107] Methods for the production of single-chain antibodies are well known
to those
of skill in the art. The skilled artisan is referred to U.S. Pat. No.
5,359,046, (incorporated
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herein by reference) for such methods. A single chain antibody is created by
fusing together
the variable domains of the heavy and light chains using a short peptide
linker, thereby
reconstituting an antigen binding site on a single molecule. Single-chain
antibody variable
fragments (scFvs) in which the C-terminus of one variable domain is tethered
to the N-
tenninus of the other variable domain via a 15 to 25 amino acid peptide or
linker have been
developed without significantly disrupting antigen binding or specificity of
the binding
(Bedzyk et al., 1990; Chaudhary et al., 1990). The linker is chosen to permit
the heavy chain
and light chain to bind together in their proper conformational orientation.
See, for example,
Huston, J. S., et al., Methods in Enzym. 203:46-121 (1991), which is
incorporated herein by
reference. These Fvs lack the constant regions (Fc) present in the heavy and
light chains of
the native antibody.
iii. Monovalent antibodies
[000108] In vitro methods are also suitable for preparing monovalent
antibodies.
Digestion of antibodies to produce fragments thereof, particularly, Fab
fragments, can be
accomplished using routine techniques known in the art. For instance,
digestion can be
performed using papain. Examples of papain digestion are described in WO
94/29348
published Dec. 22, 1994, U.S. Pat. No. 4,342,566, and Harlow and Lane,
Antibodies, A
Laboratory Manual, Cold Spring Harbor Publications, New York, (1988). Papain
digestion
of antibodies typically produces two identical antigen binding fragments,
called Fab
fragments, each with a single antigen binding site, and a residual Fc
fragment. Pepsin
treatment yields a fragment, called the F(ab')2 fragment, that has two antigen
combining
sites and is still capable of cross-linking antigen.
[000109] The Fab fragments produced in the antibody digestion also contain the
constant
domains of the light chain and the first constant domain of the heavy chain.
Fab' fragments
differ from Fab fragments by the addition of a few residues at the carboxy
terminus of the
heavy chain domain including one or more cysteines from the antibody hinge
region. The
F(ab')2 fragment is a bivalent fragment comprising two Fab' fragments linked
by a
disulfide bridge at the hinge region. Fab'-SH is the designation herein for
Fab' in which the
cysteine residue(s) of the constant domains bear a free thiol group. Antibody
fragments
originally were produced as pairs of Fab' fragments which have hinge cysteines
between
them. Other chemical couplings of antibody fragments are also known.
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iv. Chimeric/Hybrid
[000110] In hybrid antibodies, one heavy and light chain pair is homologous to
that found
in an antibody raised against one antigen recognition feature, e.g., epitope,
while the other
heavy and light chain pair is homologous to a pair found in an antibody raised
against
another epitope. This results in the property of multi-functional valency,
i.e., ability to bind
at least two different epitopes simultaneously. As used herein, the term
"hybrid antibody"
refers to an antibody wherein each chain is separately homologous with
reference to a
mammalian antibody chain, but the combination represents a novel assembly so
that two
different antigens are recognized by the antibody. Such hybrids can be formed
by fusion of
hybridomas producing the respective component antibodies, or by recombinant
techniques.
Such hybrids may, of course, also be formed using chimeric chains.
v. Anti-idiotypic
[0001111 The encoded antibodies can be anti-idiotypic antibodies (antibodies
that bind
other antibodies) as described, for example, in U.S. Pat. No. 4,699,880. Such
anti-idiotypic
antibodies could bind endogenous or foreign antibodies in a treated
individual, thereby to
ameliorate or prevent pathological conditions associated with an immune
response, e.g.; in
the context of an autoimmune disease.
vi. Conjugates or Fusions of antibody fragments
[000112] The targeting function of the antibody can be used therapeutically by
coupling
the antibody or a fragment thereof with a therapeutic agent. Such coupling of
the antibody
or fragment (e.g., at least a portion of an immunoglobulin constant region
(Fc)) with the
therapeutic agent can be achieved by making an immunoconjugate or by making a
fusion
protein, comprising the antibody or antibody fragment and the therapeutic
agent.
[000113] Also included within the meaning of "antibody or fragments thereof'
are
conjugates of antibody fragments and antigen binding proteins (single chain
antibodies) as
described, for example, in U.S. Pat. No. 4,704,692, the contents of which are
hereby
incorporated by reference. In some aspects, the antibody does not comprise an
immunoglobulin variable region but instead is a fusion protein comprising an
immunogolublin Fc region and a binding region of a ligand or receptors.
10001141 An antibody (or fragment thereof) may be conjugated to a therapeutic
moiety
such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A
cytotoxin or cytotoxic
agent includes any agent that is detrimental to cells. Examples include taxol,
cytochalasin B,
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gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide,
vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,
mitoxantrone,
mithramycin, actinomycin D, 1- dehydrotestosterone, glucocorticoids, procaine,
tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs thereof.
Therapeutic agents
include, but are not limited to, antimetabolites (e.g., methotrexate, 6-
mercaptopurine, 6-
thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,
mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and
lomustine
(CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin
C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e. g.,
daunorubicin
(formerly daunomycin) and doxorubicin), antibiotics (e.g. , dactinomycin
(formerly
actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic
agents
(e.g., vincristine and vinblastine).
[000115] The conjugates disclosed can be used for modifying a given biological
response.
The drug moiety is not to be construed as limited to classical chemical
therapeutic agents.
For example, the drug moiety may be a protein or polypeptide possessing a
desired
biological activity. Such proteins may include, for example, a toxin such as
abrin, ricin A,
pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis
factor, [agr]-
interferon, [bgr]-interferon, nerve growth factor, platelet derived growth
factor, tissue
plasminogen activator; or, biological response modifiers such as, for example,
lymphokines,
interleukin-1 ("IL-1 "), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"),
granulocyte
macrophage colony stimulating factor ("GM-CSF"), granulocyte colony
stimulating factor
("G-CSF"), or other growth factors.
[000116] Techniques for conjugating such therapeutic moiety to antibodies are
well
known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of
Drugs In
Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al.
(eds.), pp.
243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug
Delivery", in
Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel
Dekker, Inc.
1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in
Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et
al. (eds.), pp.
475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic
Use Of
Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer
Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press
1985), and
Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin
Conjugates",
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Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can be conjugated
to a second
antibody to form an antibody heteroconjugate as described by Segal in U.S.
Pat. No. 4,676,
980.
vii. Method of Making Antibodies Using Protein Chemistry
[000117] One method of producing proteins comprising the antibodies is to link
two or
more peptides or polypeptides together by protein chemistry techniques. For
example,
peptides or polypeptides can be chemically synthesized using currently
available laboratory
equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert -
butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, CA). One
skilled in
the art can readily appreciate that a peptide or polypeptide corresponding to
the antibody,
for example, can be synthesized by standard chemical reactions. For example, a
peptide or
polypeptide can be synthesized and not cleaved from its synthesis resin
whereas the other
fragment of an antibody can be synthesized and subsequently cleaved from the
resin,
thereby exposing a terminal group which is functionally blocked on the other
fragment. By
peptide condensation reactions, these two fragments can be covalently joined
via a peptide
bond at their carboxyl and amino termini, respectively, to form an antibody,
or fragment
thereof. (Grant GA (1992) Synthetic Peptides: A User Guide. W.H. Freeman and
Co., N.Y.
(1992); Bodansky M and Trost B., Ed. (1993) Principles of Peptide Synthesis.
Springer-
Verlag Inc., NY. Alternatively, the peptide or polypeptide is independently
synthesized in
vivo as described above. Once isolated, these independent peptides or
polypeptides may be
linked to form an antibody or fragment thereof via similar peptide
condensation reactions.
[000118] For example, enzymatic ligation of cloned or synthetic peptide
segments allow
relatively short peptide fragments to be joined to produce larger peptide
fragments,
polypeptides or whole protein domains (Abrahmsen L et al., Biochemistry,
30:4151 (1991)).
Alternatively, native chemical ligation of synthetic peptides can be utilized
to synthetically
construct large peptides or polypeptides from shorter peptide fragments. This
method
consists of a two step chemical reaction (Dawson et al. Synthesis of Proteins
by Native
Chemical Ligation. Science, 266:776-779 (1994)). The first step is the
chemoselective
reaction of an unprotected synthetic peptide-alpha-thioester with another
unprotected
peptide segment containing an amino-terminal Cys residue to give a thioester-
linked
intermediate as the initial covalent product. Without a change in the reaction
conditions,
this intermediate undergoes spontaneous, rapid intramolecular reaction to form
a native
peptide bond at the ligation site. Application of this native chemical
ligation method to the
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total synthesis of a protein molecule is illustrated by the preparation of
human interleukin 8
(IL-8) (Baggiolini M et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis I et
al.,
J.Biol.Chem., 269:16075 (1994); Clark-Lewis I et al., Biochemistry, 30:3128
(1991);
Rajarathnam K et al., Biochemistry 33:6623-30 (1994)).
viii. Human and Humanized
[000119] Transgenic animals (e.g., mice) that are capable, upon immunization,
of
producing a full repertoire of human antibodies in the absence of endogenous
immunoglobulin production can be employed. For example, it has been described
that the
homozygous deletion of the antibody heavy chain joining region (J(H)) gene in
chimeric
and germ-line mutant mice results in complete inhibition of endogenous
antibody
production. Transfer of the human germ-line immunoglobulin gene array in such
germ-line
mutant mice will result in the production of human antibodies upon antigen
challenge (see,
e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551-255 (1993);
Jakobovits et al.,
Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993)).
Human
antibodies can also be produced in phage display libraries (Hoogenboom et al.,
J. Mol.
Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). The
techniques of Cote
et al. and Boemer et al. are also available for the preparation of human
monoclonal
antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, p. 77
(1985); Boemer et al., J. Immunol., 147(1):86-95 (1991)).
[000120] Optionally, the antibodies are generated in other species and
"humanized" for
administration in humans. Humanized forms of non-human (e.g., murine)
antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as
Fv, Fab,
Fab', F(ab')2, or other antigen-binding subsequences of antibodies) which
contain minimal
sequence derived from non-human immunoglobulin. Humanized antibodies include
human
immunoglobulins (recipient antibody) in which residues from a complementarity
determining region (CDR) of the recipient antibody are replaced by residues
from a CDR of
a non-human species (donor antibody) such as mouse, rat or rabbit having the
desired
specificity, affinity and capacity. In some instances, Fv framework residues
of the human
immunoglobulin are replaced by corresponding non-human residues. Humanized
antibodies may also comprise residues that are found neither in the recipient
antibody nor in
the imported CDR or framework sequences. In general, the humanized antibody
will
comprise substantially all of at least one, and typically two, variable
domains, in which all
or substantially all of the CDR regions correspond to those of a non-human
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immunoglobulin and all or substantially all of the FR regions are those of a
human
immunoglobulin consensus sequence. The humanized antibody optimally also will
comprise at least a portion of an immunoglobulin constant region (Fc),
typically that of a
human immunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann et
al.,
Nature, 332:323-327 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596
(1992))
10001211 Methods for humanizing non-human antibodies are well known in the
art.
Generally, a humanized antibody has one or more amino acid residues introduced
into it
from a source that is non-human. These non-human amino acid residues are often
referred
to as "import" residues, which are typically taken from an "import" variable
domain.
Antibody humanization techniques generally involve the use of recombinant DNA
technology to manipulate the DNA sequence encoding one or more polypeptide
chains of an
antibody molecule. Humanization can be essentially performed following the
method of
Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et
al., Nature,
332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by
substituting
rodent CDRs or CDR sequences for the corresponding sequences of a human
antibody.
Accordingly, a humanized form of a non-human antibody (or a fragment thereof)
is a
chimeric antibody or fragment (U.S. Pat. No. 4,816,567), wherein substantially
less than an
intact human variable domain has been substituted by the corresponding
sequence from a
non-human species. In practice, humanized antibodies are typically human
antibodies in
which some CDR residues and possibly some FR residues are substituted by
residues from
analogous sites in rodent antibodies.
[000122] The choice of human variable domains, both light and heavy, to be
used in
making the humanized antibodies is very important in order to reduce
antigenicity.
According to the "best-fit" method, the sequence of the variable domain of a
rodent
antibody is screened against the entire library of known human variable domain
sequences.
The human sequence which is closest to that of the rodent is then accepted as
the human
framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151:2296
(1993) and
Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses a
particular framework
derived from the consensus sequence of all human antibodies of a particular
subgroup of
light or heavy chains. The same framework may be used for several different
humanized
antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta
et al., J.
Immunol., 151:2623 (1993)).
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[0001231 It is further important that antibodies be humanized with retention
of high
affinity for the antigen and other favorable biological properties. To achieve
this goal,
according to a preferred method, humanized antibodies are prepared by a
process of
analysis of the parental sequences and various conceptual humanized products
using three
dimensional models of the parental and humanized sequences. Three dimensional
immunoglobulin models are commonly available and are familiar to those skilled
in the art.
Computer programs are available which illustrate and display probable three-
dimensional
conformational structures of selected candidate immunoglobulin sequences.
Inspection of
these displays permits analysis of the likely role of the residues in the
functioning of the
candidate immunoglobulin sequence, i.e., the analysis of residues that
influence the ability
of the candidate immunoglobulin to bind its antigen. In this way, FR residues
can be
selected and combined from the consensus and import sequence so that the
desired antibody
characteristic, such as increased affinity for the target antigen(s), is
achieved. In general,
the CDR residues are directly and most substantially involved in influencing
antigen
binding (see, WO 94/04679, published 3 March 1994).
[000124] As used herein, the term "epitope" is meant to include any
determinant capable
of specific interaction with the antibodies disclosed. Epitopic determinants
usually consist
of chemically active surface groupings of molecules such as amino acids or
sugar side
chains and usually have specific three dimensional structural characteristics,
as well as
specific charge characteristics.
[000125] An "epitope tag" denotes a short peptide sequence unrelated to the
function of
the antibody or molecule that can be used for purification or crosslinking of
the molecule
with anti-epitope tag antibodies or other reagents.
[000126] By "specifically binds" is meant that an antibody recognizes and
physically
interacts with its cognate antigen and does not significantly recognize and
interact with
other antigens; such an antibody may be a polyclonal antibody or a monoclonal
antibody,
which are generated by techniques that are well known in the art.
[000127] The antibody can be bound to a substrate or labeled with a detectable
moiety or
both bound and labeled. The detectable moieties contemplated with the present
compositions include fluorescent, enzymatic and radioactive markers.
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ix. Administration of antibodies
[000128] Administration of the antibodies can be done as disclosed herein.
Nucleic acid
approaches for antibody delivery also exist. The broadly neutralizing
antibodies and
antibody fragments can also be administered to patients or subjects as a
nucleic acid
preparation (e.g., DNA or RNA) that encodes the antibody or antibody fragment,
such that
the patient's or subject's own cells take up the nucleic acid and produce and
secrete the
encoded antibody or antibody fragment. The delivery of the nucleic acid can be
by any
means, as disclosed herein, for example.
[000129] The nucleic acid of the disclosed compositions and methods can be any
nucleic
acid. For example, the nucleic acid can be a functional nucleic acid or a
nucleic acid
encoding a therapeutic protein or peptide. Thus, the disclosed composition can
comprise a
vector comprising a nucleic acid, wherein the nucleic acid is a functional
nucleic acid or a
nucleic acid that encodes a therapeutic protein or peptide. The disclosed
nucleic acids can
be made up of for example, nucleotides, nucleotide analogs, or nucleotide
substitutes. Non-
limiting examples of these and other molecules are discussed herein. It is
understood that
for example, when a vector is expressed in a cell, the expressed mRNA will
typically be
made up of A, C, G, and U. Likewise, it is understood that if, for example, an
antisense
molecule is introduced into a cell or cell environment through for example
exogenous
delivery, it is advantageous that the antisense molecule be made up of
nucleotide analogs
that reduce the degradation of the antisense molecule in the cellular
environment.
[000130] A nucleotide is a molecule that contains a base moiety, a sugar
moiety and a
phosphate moiety. Nucleotides can be linked together through their phosphate
moieties and
sugar moieties creating an intemucleoside linkage. The base moiety of a
nucleotide can be
adenin-9-yl (A), cytosin-l-yl (C), guanin-9-yl (G), uracil-l-yl (U), and
thymin-l-yl (T).
The sugar moiety of a nucleotide is a ribose or a deoxyribose. The phosphate
moiety of a
nucleotide is pentavalent phosphate. A non-limiting example of a nucleotide
would be 3'-
AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate). There
are
many varieties of these types of molecules available in the art and available
herein.
[000131] A nucleotide analog is a nucleotide which contains some type of
modification to
either the base, sugar, or phosphate moieties. Modifications to nucleotides
are well known
in the art and would include for example, 5-methylcytosine (5-me-C), 5-
hydroxymethyl
cytosine, xanthine, hypoxanthine, and 2-aminoadenine as well as modifications
at the sugar
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or phosphate moieties. There are many varieties of these types of molecules
available in the
art and available herein.
[000132] Nucleotide substitutes are molecules having similar functional
properties to
nucleotides, but which do not contain a phosphate moiety, such as peptide
nucleic acid
(PNA). Nucleotide substitutes are molecules that will recognize nucleic acids
in a Watson-
Crick or Hoogsteen manner, but which are linked together through a moiety
other than a
phosphate moiety. Nucleotide substitutes are able to conform to a double helix
type
structure when interacting with the appropriate target nucleic acid. There are
many varieties
of these types of molecules available in the art and available herein.
[000133] It is also possible to link other types of molecules (conjugates) to
nucleotides or
nucleotide analogs to enhance for example, cellular uptake. Conjugates can be
chemically
linked to the nucleotide or nucleotide analogs. Such conjugates include but
are not limited
to lipid moieties such as a cholesterol moiety. (Letsinger et al., Proc. Natl.
Acad. Sci. USA,
1989,86, 6553-6556). There are many varieties of these types of molecules
available in the
art and available herein.
[000134] A Watson-Crick interaction is at least one interaction with the
Watson-Crick
face of a nucleotide, nucleotide analog, or nucleotide substitute. The Watson-
Crick face of
a nucleotide, nucleotide analog, or nucleotide substitute includes the C2, Nl,
and C6
positions of a purine based nucleotide, nucleotide analog, or nucleotide
substitute and the
C2, N3, C4 positions of a pyrimidine based nucleotide, nucleotide analog, or
nucleotide
substitute.
[000135] A Hoogsteen interaction is the interaction that takes place on the
Hoogsteen
face of a nucleotide or nucleotide analog, which is exposed in the major
groove of duplex
DNA. The Hoogsteen face includes the N7 position and reactive groups (NH2 or
0) at the
C6 position of purine nucleotides.
i. Functional Nucleic Acids
[000136] Functional nucleic acids are nucleic acid molecules that have a
specific
function, such as binding a target molecule or catalyzing a specific reaction.
Functional
nucleic acid molecules can be divided into the following categories, which are
not meant to
be limiting. For example, functional nucleic acids include antisense
molecules, aptamers,
ribozymes, triplex forming molecules, RNAi, and external guide sequences. The
functional
nucleic acid molecules can act as affectors, inhibitors, modulators, and
stimulators of a
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specific activity possessed by a target molecule, or the functional nucleic
acid molecules
can possess a de novo activity independent of any other molecules.
[000137] Functional nucleic acid molecules can interact with any
macromolecule, such as
DNA, RNA, polypeptides, or carbohydrate chains. Often functional nucleic acids
are
designed to interact with other nucleic acids based on sequence homology
between the
target molecule and the functional nucleic acid molecule. In other situations,
the specific
recognition between the functional nucleic acid molecule and the target
molecule is not
based on sequence homology between the functional nucleic acid molecule and
the target
molecule, but rather is based on the formation of tertiary structure that
allows specific
recognition to take place.
[000138] Antisense molecules are designed to interact with a target nucleic
acid molecule
through either canonical or non-canonical base pairing. The interaction of the
antisense
molecule and the target molecule is designed to promote the destruction of the
target
molecule through, for example, RNAseH mediated RNA-DNA hybrid degradation.
Alternatively the antisense molecule is designed to interrupt a processing
function that
normally would take place on the target molecule, such as transcription or
replication.
Antisense molecules can be designed based on the sequence of the target
molecule.
Numerous methods for optimization of antisense efficiency by finding the most
accessible
regions of the target molecule exist. Exemplary methods would be in vitro
selection
experiments and DNA modification studies using DMS and DEPC. It is preferred
that
antisense molecules bind the target molecule with a dissociation constant
(Kd)less than or
equal to 10-6, 10, 100, or 10-12 . A representative sample of methods and
techniques which
aid in the design and use of antisense molecules can be found in U.S. Patent
Nos. 5,135,917,
5,294,533, 5,627,158, 5,641,754, 5,691,317, 5,780,607, 5,786,138, 5,849,903,
5,856,103,
5,919,772, 5,955,590, 5,990,088, 5,994,320, 5,998,602, 6,005,095, 6,007,995,
6,013,522,
6,017,898, 6,018,042, 6,025,198, 6,033,910, 6,040,296, 6,046,004, 6,046,319,
and
6,057,437.
[000139] Aptamers are molecules that interact with a target molecule,
preferably in a
specific way. Typically aptamers are small nucleic acids ranging from 15-50
bases in
length that fold into defined secondary and tertiary structures, such as stem-
loops or G-
quartets. Aptamers can bind small molecules, such as ATP (U.S. Patent No.
5,631,146) and
theophiline (U.S. Patent No. 5,580,737), as well as large molecules, such as
reverse
transcriptase (U.S. Patent No. 5,786,462) and thrombin (United States patent
5,543,293).
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Aptamers can bind very tightly with Kd's from the target molecule of less than
10-12 M. It
is preferred that the aptamers bind the target molecule with a Kd less than
10"6, 10-8, 10-10, or
10-12. Aptamers can bind the target molecule with a very high degree of
specificity. For
example, aptamers have been isolated that have greater than a 10,000 fold
difference in
binding affinities between the target molecule and another molecule that
differ at only a
single position on the molecule (U.S. Patent No. 5,543,293). It is preferred
that the aptamer
have a Kd with the target molecule at least 10, 100, 1000, 10,000, or 100,000
fold lower
than the Kd with a background binding molecule. It is preferred when doing the
comparison
for a polypeptide for example, that the background molecule be a different
polypeptide.
Representative examples of how to make and use aptamers to bind a variety of
different
target molecules can be found in U.S. Patent Nos. 5,476,766, 5,503,978,
5,631,146,
5,731,424 , 5,780,228, 5,792,613, 5,795,721, 5,846,713, 5,858,660, 5,861,254,
5,864,026,
5,869,641, 5,958,691, 6,001,988, 6,011,020, 6,013,443, 6,020,130, 6,028,186,
6,030,776,
and 6,051,698.
[000140] Ribozymes are nucleic acid molecules that are capable of catalyzing a
chemical
reaction, either intramolecularly or intermolecularly. Ribozymes are thus
catalytic nucleic
acid. It is preferred that the ribozymes catalyze intermolecular reactions.
There are a
number of different types of ribozymes that catalyze nuclease or nucleic acid
polymerase
type reactions which are based on ribozymes found in natural systems, such as
hammerhead
ribozymes, (U.S. Patent Nos. 5,334,711, 5,436,330, 5,616,466, 5,633,133,
5,646,020,
5,652,094, 5,712,384, 5,770,715, 5,856,463, 5,861,288, 5,891,683, 5,891,684,
5,985,621,
5,989,908, 5,998,193, 5,998,203; International Patent Application Nos. WO
9858058 by
Ludwig and Sproat, WO 9858057 by Ludwig and Sproat, and WO 9718312 by Ludwig
and
Sproat) hairpin ribozymes (for example, U.S. Patent Nos. 5,631,115, 5,646,031,
5,683,902,
5,712,384, 5,856,188, 5,866,701, 5,869,339, and 6,022,962), and tetrahymena
ribozymes
(for example, U.S. Patent Nos. 5,595,873 and 5,652,107). There are also a
number of
ribozymes that are not found in natural systems, but which have been
engineered to catalyze
specific reactions de novo (for example, U.S. Patent Nos. 5,580,967,
5,688,670, 5,807,718,
and 5,910,408). Preferred ribozymes cleave RNA or DNA substrates, and more
preferably
cleave RNA substrates. Ribozymes typically cleave nucleic acid substrates
through
recognition and binding of the target substrate with subsequent cleavage. This
recognition
is often based mostly on canonical or non-canonical base pair interactions.
This property
makes ribozymes particularly good candidates for target specific cleavage of
nucleic acids
because recognition of the target substrate is based on the target substrates
sequence.
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Representative examples of how to make and use ribozymes to catalyze a variety
of
different reactions can be found in U.S. Patent Nos. 5,646,042, 5,693,535,
5,731,295,
5,811,300, 5,837,855, 5,869,253, 5,877,021, 5,877,022, 5,972,699, 5,972,704,
5,989,906,
and 6,017,756.
[000141] Triplex forming functional nucleic acid molecules are molecules that
can
interact with either double-stranded or single-stranded nucleic acid. When
triplex
molecules interact with a target region, a structure called a triplex is
formed, in which there
are three strands of DNA forming a complex dependant on both Watson-Crick and
Hoogsteen base-pairing. Triplex molecules are preferred because they can bind
target
regions with high affinity and specificity. It is preferred that the triplex
forming molecules
bind the target molecule with a Kd less than 10, 10-g, 10-10, or 10-12.
Representative
examples of how to make and use triplex forming molecules to bind a variety of
different
target molecules can be found in U.S. Patent Nos. 5,176,996, 5,645,985,
5,650,316,
5,683,874, 5,693,773, 5,834,185, 5,869,246, 5,874,566, and 5,962,426.
[000142] External guide sequences (EGSs) are molecules that bind a target
nucleic acid
molecule forming a complex, and this complex is recognized by RNase P, which
cleaves the
target molecule. EGSs can be designed to specifically target a RNA molecule of
choice.
RNAse P aids in processing transfer RNA (tRNA) within a cell. Bacterial RNAse
P can be
recruited to cleave virtually any RNA sequence by using an EGS that causes the
target
RNA:EGS complex to mimic the natural tRNA substrate. (WO 92/03566 by Yale, and
Forster and Altman, Science 238:407-409 (1990)).
[000143] Similarly, eukaryotic EGS/RNAse P-directed cleavage of RNA can be
utilized
to cleave desired targets within eukarotic cells. (Yuan et al., Proc. Natl.
Acad. Sci. USA
89:8006-8010 (1992); WO 93/22434 by Yale; WO 95/24489 by Yale; Yuan and
Altman,
EMBO J 14:159-168 (1995), and Carrara et al., Proc. Natl. Acad. Sci. (USA)
92:2627-2631
(1995)). Representative examples of how to make and use EGS molecules to
facilitate
cleavage of a variety of different target molecules be found in U.S. Patent
Nos. 5,168,053,
5,624,824, 5,683,873, 5,728,521, 5,869,248, and 5,877,162.
[000144] Gene expression can also be effectively silenced in a highly specific
manner
through RNA interference (RNAi). This silencing was originally observed with
the addition
of double stranded RNA (dsRNA) (Fire,A., et al. (1998) Nature, 391:806-11;
Napoli, C., et
al. (1990) Plant Cel12:279-89; Hannon, G.J. (2002) Nature, 418:244-51). Once
dsRNA
enters a cell, it is cleaved by an RNase III -like enzyme, Dicer, into double
stranded small
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interfering RNAs (siRNA) 21-23 nucleotides in length that contains 2
nucleotide overhangs
on the 3' ends (Elbashir, S.M., et al. (2001) Genes Dev., 15:188-200;
Bernstein, E., et al.
(2001) Nature, 409:363-6; Hammond, S.M., et al. (2000) Nature, 404:293-6). In
an ATP
dependent step, the siRNAs become integrated into a multi-subunit protein
complex,
commonly known as the RNAi induced silencing complex (RISC), which guides the
siRNAs to the target RNA sequence (Nykanen, A., et al. (2001) Cell, 107:309-
21). At some
point the siRNA duplex unwinds, and it appears that the antisense strand
remains bound to
RISC and directs degradation of the complementary mRNA sequence by a
combination of
endo and exonucleases (Martinez, J., et al. (2002) Cell, 110:563-74). However,
the effect of
iRNA or siRNA or their use is not limited to any type of mechanism.
[000145] Short Interfering RNA (siRNA) is a double-stranded RNA that can
induce
sequence-specific post-transcriptional gene silencing, thereby decreasing or
even inhibiting
gene expression. In one example, an siRNA triggers the specific degradation of
homologous
RNA molecules, such as mRNAs, within the region of sequence identity between
both the
siRNA and the target RNA. For example, WO 02/44321 discloses siRNAs capable of
sequence-specific degradation of target mRNAs when base-paired with 3'
overhanging
ends, herein incorporated by reference for the method of making these siRNAs.
Sequence
specific gene silencing can be achieved in mammalian cells using synthetic,
short double-
stranded RNAs that mimic the siRNAs produced by the enzyme dicer (Elbashir,
S.M., et al.
(2001) Nature, 411:494 498) (Ui-Tei, K., et al. (2000) FEBS Lett 479:79-82).
siRNA can be
chemically or in vitro-synthesized or can be the result of short double-
stranded hairpin-like
RNAs (shRNAs) that are processed into siRNAs inside the cell. Synthetic siRNAs
are
generally designed using algorithms and a conventional DNA/RNA synthesizer.
Suppliers
include Ambion (Austin, Texas), ChemGenes (Ashland, Massachusetts), Dharmacon
(Lafayette, Colorado), Glen Research (Sterling, Virginia), MWB Biotech
(Esbersberg,
Germany), Proligo (Boulder, Colorado), and Qiagen (Vento, The Netherlands).
siRNA can
also be synthesized in vitro using kits such as Ambion's SILENCER siRNA
Construction
Kit.
[000146] The production of siRNA from a vector is more commonly done through
the
transcription of a short hairpin RNAs (shRNAs). Kits for the production of
vectors
comprising shRNA are available, such as, for example, Imgenex's
GENESUPPRESSORTM
Construction Kits and Invitrogen's BLOCK-ITTM inducible RNAi plasmid and
lentivirus
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vectors. Disclosed herein are any shRNA designed as described above based on
the
sequences for the herein disclosed inflammatory mediators.
ii. Nucleic Acid Vector
[000147] There are a number of compositions and methods which can be used to
deliver
nucleic acids, such as nucleic acids encoding therapeutic proteins and
peptides, to cells,
either in vitro or in vivo. These methods and compositions can largely be
broken down into
two classes: viral based delivery systems and non-viral based delivery
systems. For
example, the nucleic acids can be delivered through a number of direct
delivery systems
such as, electroporation, lipofection, calcium phosphate precipitation,
plasmids, viral
vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via
transfer of genetic
material in cells or carriers such as cationic liposomes. Appropriate means
for transfection,
including viral vectors, chemical transfectants, or physico-mechanical methods
such as
electroporation and direct diffusion of DNA, are described by, for example,
Wolff, J. A., et
al., Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818,
(1991)Such
methods are well known in the art and readily adaptable for use with the
compositions and
methods described herein. In certain cases, the methods can be modified to
specifically
function with large DNA molecules. Further, these methods can be used to
target certain
diseases and cell populations by using the targeting characteristics of the
carrier.
a. Nucleic acid based delivery systems
[000148] Transfer vectors can be any nucleotide construction used to deliver
genes into
cells (e.g., a plasmid), or as part of a general strategy to deliver genes,
e.g., as part of
recombinant retrovirus or adenovirus (Ram et al. Cancer Res. 53:83-88,
(1993)).
[000149] As used herein, plasmid or viral vectors are agents that transport
nucleic acids
into a cell without degradation and include a promoter yielding expression of
the gene in the
cells into which it is delivered. Viral vectors are, for example, Adenovirus,
Adeno-
associated virus, Herpes virus, Vaccinia virus, Polio virus, AIDS virus,
neuronal trophic
virus, Sindbis and other RNA viruses, including these viruses with the HIV
backbone. Also
preferred are any viral families which share the properties of these viruses
which make them
suitable for use as vectors. Retroviruses include Murine Maloney Leukemia
virus, MMLV,
and retroviruses that express the desirable properties of MMLV as a vector.
Retroviral
vectors are able to carry a larger genetic payload, i.e., a transgene or
marker gene, than other
viral vectors, and for this ireason are a commonly used vector. However, they
are not as
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useful in non-proliferating cells. Adenovirus vectors are relatively stable
and easy to work
with, have high titers, and can be delivered in aerosol formulation, and can
transfect non-
dividing cells. Pox viral vectors are large and have several sites for
inserting genes, they are
thermostable and can be stored at room temperature. A preferred embodiment is
a viral
vector which has been engineered so as to suppress the immune response of the
host
organism, elicited by the viral antigens. Preferred vectors of this type will
carry coding
regions for Interleukin 8 or 10.
[000150] Viral vectors can have higher transaction (ability to introduce
genes) abilities
than chemical or physical methods to introduce genes into cells. Typically,
viral vectors
contain, nonstructural early genes, structural late genes, an RNA polymerase
III transcript,
inverted terminal repeats necessary for replication and encapsidation, and
promoters to
control the transcription and replication of the viral genome. When engineered
as vectors,
viruses typically have one or more of the early genes removed and a gene or
gene/promotor
cassette is inserted into the viral genome in place of the removed viral DNA.
Constructs of
this type can carry up to about 8 kb of foreign genetic material. The
necessary functions of
the removed early genes are typically supplied by cell lines which have been
engineered to
express the gene products of the early genes in trans.
(A) Retroviral Vectors
[000151] A retrovirus is an animal virus belonging to the virus family of
Retroviridae,
including any types, subfamilies, genus, or tropisms. Retroviral vectors, in
general, are
described by Verma, I.M., Retroviral vectors for gene transfer. In
Microbiology-1985,
American Society for Microbiology, pp. 229-232, Washington, (1985), which is
incorporated by reference herein. Examples of methods for using retroviral
vectors for gene
therapy are described in U.S. Patent Nos. 4,868,116 and 4,980,286; PCT
applications WO
90/02806 and WO 89/07136; and Mulligan, (Science 260:926-932 (1993)); the
teachings of
which are incorporated herein by reference.
[000152] A retrovirus is essentially a package which has packed into it
nucleic acid cargo.
The nucleic acid cargo carries with it a packaging signal, which ensures that
the replicated
daughter molecules will be efficiently packaged within the package coat. In
addition to the
package signal, there are a number of molecules which are needed in cis, for
the replication,
and packaging of the replicated virus. Typically a retroviral genome, contains
the gag, pol,
and env genes which are involved in the making of the protein coat. It is the
gag, pol, and
env genes which are typically replaced by the foreign DNA that it is to be
transferred to the
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target cell. Retrovirus vectors typically contain a packaging signal for
incorporation into
the package coat, a sequence which signals the start of the gag transcription
unit, elements
necessary for reverse transcription, including a primer binding site to bind
the tRNA primer
of reverse transcription, terminal repeat sequences that guide the switch of
RNA strands
during DNA synthesis, a purine rich sequence 5' to the 3' LTR that serve as
the priming site
for the synthesis of the second strand of DNA synthesis, and specific
sequences near the
ends of the LTRs that enable the insertion of the DNA state of the retrovirus
to insert into
the host genome. The removal of the gag, pol, and env genes allows for about 8
kb of
foreign sequence to be inserted into the viral genome, become reverse
transcribed, and upon
replication be packaged into a new retroviral particle. This amount of nucleic
acid is
sufficient for the delivery of a one to many genes depending on the size of
each transcript.
It is preferable to include either positive or negative selectable markers
along with other
genes in the insert.
[000153] Since the replication machinery and packaging proteins in most
retroviral
vectors have been removed (gag, pol, and env), the vectors are typically
generated by
placing them into a packaging cell line. A packaging cell line is a cell line
which has been
transfected or transformed with a retrovirus that contains the replication and
packaging
machinery, but lacks any packaging signal. When the vector carrying the DNA of
choice is
transfected into these cell lines, the vector containing the gene of interest
is replicated and
packaged into new retroviral particles, by the machinery provided in cis by
the helper cell.
The genomes for the machinery are not packaged because they lack the necessary
signals.
(B) Adenoviral Vectors
[000154] The construction of replication-defective adenoviruses has been
described
(Berkner et al., J. Virology 61:1213-1220 (1987); Massie et al., Mol. Cell.
Biol. 6:2872-
2883 (1986); Haj-Ahmad et al., J. Virology 57:267-274 (1986); Davidson et al.,
J.
Virology 61:1226-1239 (1987); Zhang "Generation and identification of
recombinant
adenovirus by liposome-mediated transfection and PCR analysis" BioTechniques
15:868-
872 (1993)). The benefit of the use of these viruses as vectors is that they
are limited in the
extent to which they can spread to other cell types, since they can replicate
within an initial
infected cell, but are unable to fonn new infectious viral particles.
Recombinant
adenoviruses have been shown to achieve high efficiency gene transfer after
direct, in vivo
delivery to airway epithelium, hepatocytes, vascular endothelium, CNS
parenchyma and a
number of other tissue sites (Morsy, J. Clin. Invest. 92:1580-1586 (1993);
Kirshenbaum,
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J. Clin. Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092
(1993);
Moullier, Nature Genetics 4:154-159 (1993); La Salle, Science 259:988-990
(1993);
Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992); Rich, Human Gene Therapy
4:461-
476 (1993); Zabner, Nature Genetics 6:75-83 (1994); Guzman, Circulation
Research
73:1201-1207 (1993); Bout, Human Gene Therapy 5:3-10 (1994); Zabner,
Cel175:207-
216 (1993); Caillaud, Eur. J. Neuroscience 5:1287-1291 (1993); and Ragot, J.
Gen.
Virology 74:501-507 (1993)). Recombinant adenoviruses achieve gene
transduction by
binding to specific cell surface receptors, after which the virus is
internalized by receptor-
mediated endocytosis, in the same manner as wild type or replication-defective
adenovirus
(Chardonnet and Dales, Virology 40:462-477 (1970); Brown and Burlingham, J.
Virology
12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985); Seth,
et al., J.
Virol. 51:650-655 (1984); Seth, et al., Mol. Cell. Biol. 4:1528-1533 (1984);
Varga et al.,
J. Virology 65:6061-6070 (1991); Wickham et al., Ce1173:309-319 (1993)).
[000155] A viral vector can be one based on an adenovirus which has had the El
gene
removed and these virons are generated in a cell line such as the human 293
cell line. In
another preferred embodiment both the E1 and E3 genes are removed from the
adenovirus
genome.
(C) Adeno-Asscociated Viral Vectors
[000156] Another type of viral vector is based on an adeno-associated virus
(AAV). This
defective parvovirus is a preferred vector because it can infect many cell
types and is
nonpathogenic to humans. AAV type vectors can transport about 4 to 5 kb and
wild type
AAV is known to stably insert into chromosome 19. Vectors which contain this
site
specific integration property are preferred. An especially preferred
embodiment of this type
of vector is the P4.1 C vector produced by Avigen, San Francisco, CA, which
can contain
the herpes simplex virus thymidine kinase gene, HSV-tk, and/or a marker gene,
such as the
gene encoding the green fluorescent protein, GFP.
[000157] In another type of AAV virus, the AAV contains a pair of inverted
terminal
repeats (ITRs) which flank at least one cassette containing a promoter which
directs cell-
specific expression operably linked to a heterologous gene. Heterologous in
this context
refers to any nucleotide sequence or gene which is not native to the AAV or B
19
parvovirus.
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[000158] Typically the AAV and B19 coding regions have been deleted, resulting
in a
safe, noncytotoxic vector. The AAV ITRs, or modifications thereof, confer
infectivity and
site-specific integration, but not cytotoxicity, and the promoter directs cell-
specific
expression. United states Patent No. 6,261,834 is herein incorproated by
reference for
material related to the AAV vector.
[000159] The disclosed vectors thus provide DNA molecules which are capable of
integration into a mammalian chromosome without substantial toxicity.
[000160] The inserted genes in viral and retroviral usually contain promoters,
and/or
enhancers to help control the expression of the desired gene product. A
promoter is
generally a sequence or sequences of DNA that function when in a relatively
fixed location
in regard to the transcription start site. A promoter contains core elements
required for
basic interaction of RNA polymerase and transcription factors, and may contain
upstream
elements and response elements.
(D) Large Payload Viral Vectors
[000161] Molecular genetic experiments with large human herpesviruses have
provided a
means whereby large heterologous DNA fragments can be cloned, propagated and
established in cells permissive for infection with herpesviruses (Sun et al.,
Nature genetics
8: 33-41, 1994; Cotter and Robertson,.Curr Opin Mol Ther 5: 633-644, 1999).
These large
DNA viruses (herpes simplex virus (HSV) and Epstein-Barr virus (EBV), have the
potential
to deliver fragments of human heterologous DNA > 150 kb to specific cells. EBV
recombinants can maintain large pieces of DNA in the infected B-cells as
episomal DNA.
Individual clones carried human genomic inserts up to 330 kb appeared
genetically stable
The maintenance of these episomes requires a specific EBV nuclear protein,
EBNA1,
constitutively expressed during infection with EBV. Additionally, these
vectors can be used
for transfection, where large amounts of protein can be generated transiently
in vitro.
Herpesvirus amplicon systems are also being used to package pieces of DNA >
220 kb and
to infect cells that can stably maintain DNA as episomes.
[000162] Other useful systems include, for example, replicating and host-
restricted non-
replicating vaccinia virus vectors.
[000163] Nucleic acids that are delivered to cells which are to be integrated
into the host
cell genome, typically contain integration sequences. These sequences are
often viral
related sequences, particularly when viral based systems are used. These viral
intergration
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systems can also be incorporated into nucleic acids which are to be delivered
using a non-
nucleic acid based system of deliver, such as a liposome, so that the nucleic
acid contained
in the delivery system can be come integrated into the host genome.
[000164] Other general techniques for integration into the host genome
include, for
example, systems designed to promote homologous recombination with the host
genome.
These systems typically rely on sequence flanking the nucleic acid to be
expressed that has
enough homology with a target sequence within the host cell genome that
recombination
between the vector nucleic acid and the target nucleic acid takes place,
causing the delivered
nucleic acid to be integrated into the host genome. These systems and the
methods
necessary to promote homologous recombination are known to those of skill in
the art.
b. Non-Nucleic Acid Based Systems
[000165] The nucleic acid of the disclosed compositions and methods can be
delivered to
the target cells in a variety of ways. For example, the compositions can be
delivered
through electroporation, or through lipofection, or through calcium phosphate
precipitation.
The delivery mechanism chosen will depend in part on the type of cell targeted
and whether
the delivery is occurring for example in vivo or in vitro.
[000166] Thus, the compositions can comprise, for example, lipids such as
liposomes,
such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic
liposomes.
Liposomes can further comprise proteins to facilitate targeting a particular
cell, if desired.
Administration of a composition comprising a compound and a cationic liposome
can be
administered to the blood afferent to a target organ or inhaled into the
respiratory tract to
target cells of the respiratory tract. Regarding liposomes, see, e.g., Brigham
et al. Am. J.
Resp. Cell. Mol. Biol. 1:95-100 (1989); Felgner et al. Proc. Natl. Acad. Sci
USA
84:7413-7417 (1987); U.S. Pat. No.4,897,355. Furthermore, the compound can be
administered as a component of a microcapsule that can be targeted to specific
cell types,
such as macrophages, or where the diffusion of the compound or delivery of the
compound
from the microcapsule is designed for a specific rate or dosage.
[000167] In the methods described above which include the administration and
uptake of
exogenous DNA into the cells of a subject (i.e., gene transduction or
transfection), delivery
of the compositions to cells can be via a variety of mechanisms. As one
example, delivery
can be via a liposome, using commercially available liposome preparations such
as
LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT
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(Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison,
WI), as well as other liposomes developed according to procedures standard in
the art. In
addition, the disclosed nucleic acid or vector can be delivered in vivo by
electroporation, the
technology for which is available from Genetronics, Inc. (San Diego, CA) as
well as by
means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, AZ).
[0001681 The materials may be in solution, suspension (for example,
incorporated into
microparticles, liposomes, or cells). These may be targeted to a particular
cell type via
antibodies, receptors, or receptor ligands. The following references are
examples of the use
of this technology to target specific proteins to tumor tissue, the principles
of which can be
applied to targeting,of other cells (Senter, et al., Bioconjugate Chem., 2:447-
451, (1991);
Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J.
Cancer,
58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993);
Battelli, et al.,
Cancer Immunol. Inimunother., 35:421-425, (1992); Pietersz and McKenzie,
Immunolog.
Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol, 42:2062-
2065,
(1991)). These techniques can be used for a variety of other specific cell
types. Vehicles
such as "stealth" and other antibody conjugated liposomes (including lipid
mediated drug
targeting to colonic carcinoma), receptor mediated targeting of DNA through
cell specific
ligands, lymphocyte directed tumor targeting, and highly specific therapeutic
retroviral
targeting of murine glioma cells in vivo. The following references are
examples of the use
of this technology to target specific proteins to tumor tissue (Hughes et al.,
Cancer
Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et
Biophysica Acta,
1104:179-187, (1992)). In general, receptors are involved in pathways of
endocytosis,
either constitutive or ligand induced. These receptors cluster in clathrin-
coated pits, enter
the cell via clathrin-coated vesicles, pass through an acidified endosome in
which the
receptors are sorted, and then either recycle to the cell surface, become
stored
intracellularly, or are degraded in lysosomes. The internalization pathways
serve a variety
of functions, such as nutrient uptake, removal of activated proteins,
clearance of
macromolecules, opportunistic entry of viruses and toxins, dissociation and
degradation of
ligand, and receptor-level regulation. Many receptors follow more than one
intracellular
pathway, depending on the cell type, receptor concentration, type of ligand,
ligand valency,
and ligand concentration. Molecular and cellular mechanisms of receptor-
mediated
endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6,
399-409
(1991)).
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[000169] Nucleic acids that are delivered to cells which are to be integrated
into the host
cell genome, typically contain integration sequences. These sequences are
often viral
related sequences, particularly when viral based systems are used. These viral
intergration
systems can also be incorporated into nucleic acids which are to be delivered
using a non-
nucleic acid based system of deliver, such as a liposome, so that the nucleic
acid contained
in the delivery system can be come integrated into the host genome.
[000170] Other general techniques for integration into the host genome
include, for
example, systems designed to promote homologous recombination with the host
genome.
These systems typically rely on sequence flanking the nucleic acid to be
expressed that has
enough homology with a target sequence within the host cell genome that
recombination
between the vector nucleic acid and the target nucleic acid takes place,
causing the delivered
nucleic acid to be integrated into the host genome. These systems and the
methods
necessary to promote homologous recombination are known to those of skill in
the art.
c. Expression Systems
[000171] The nucleic acids that are delivered to cells typically contain
expression
controlling systems. For example, the inserted genes in viral and retroviral
systems usually
contain promoters, and/or enhancers to help control the expression of the
desired gene
product. A promoter is generally a sequence or sequences of DNA that function
when in a
relatively fixed location in regard to the transcription start site. A
promoter contains core
elements required for basic interaction of RNA polymerase and transcription
factors, and
may contain upstream elements and response elements.
(A) Viral Promoters and Enhancers
[000172] Preferred promoters controlling transcription from vectors in
mammalian host
cells may be obtained from various sources, for example, the genomes of
viruses such as:
polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus
and most
preferably cytomegalovirus, or from heterologous mammalian promoters, e.g.
beta actin
promoter. The early and late promoters of the SV40 virus are conveniently
obtained as an
SV40 restriction fragment which also contains the SV40 viral origin of
replication (Fiers et
al., Nature, 273: 113 (1978)). The immediate early promoter of the human
cytomegalovirus is conveniently obtained as a HindIII E restriction fragment
(Greenway,
P.J. et al., Gene 18: 355-360 (1982)). Of course, promoters from the host cell
or related
species also are useful herein.
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[000173] Enhancer generally refers to a sequence of DNA that functions at no
fixed
distance from the transcription start site and can be either 5' (Laimins, L.
et al., Proc. Natl.
Acad. Sci. 78: 993 (1981)) or 3' (Lusky, M.L., et al., Mol. Cell Bio. 3: 1108
(1983)) to the
transcription unit. Furthermore, enhancers can be within an intron (Banerji,
J.L. et al., Cell
33: 729 (1983)) as well as within the coding sequence itself (Osborne, T.F.,
et al., Mol. Cell
Bio. 4: 1293 (1984)). They are usually between 10 and 300 bp in length, and
they function
in cis. Enhancers f unction to increase transcription from nearby promoters.
Enhancers also
often contain response elements that mediate the regulation of transcription.
Promoters can
also contain response elements that mediate the regulation of transcription.
Enhancers often
determine the regulation of expression of a gene. While many enhancer
sequences are now
known from mammalian genes (globin, elastase, albumin, a-fetoprotein and
insulin),
typically one will use an enhancer from a eukaryotic cell virus for general
expression.
Preferred examples are the SV40 enhancer on the late side of the replication
origin (bp
100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late
side of the replication origin, and adenovirus enhancers.
[000174] The promotor and/or enhancer may be specifically activated either by
light or
specific chemical events which trigger their function. Systems can be
regulated by reagents
such as tetracycline and dexamethasone. There are also ways to enhance viral
vector gene
expression by exposure to irradiation, such as gamma irradiation, or
alkylating
chemotherapy drugs.
[000175] In certain embodiments the promoter and/or enhancer region can act as
a
constitutive promoter and/or enhancer to maximize expression of the region of
the
transcription unit to be transcribed. In certain constructs the promoter
and/or enhancer
region be active in all eukaryotic cell types, even if it is only expressed in
a particular type
of cell at a particular time. A preferred promoter of this type is the CMV
promoter (650
bases). Other preferred promoters are SV40 promoters, cytomegalovirus (full
length
promoter), and retroviral vector LTR.
[000176] It has been shown that all specific regulatory elements can be cloned
and used
to construct expression vectors that are selectively expressed in specific
cell types such as
melanoma cells. The glial fibrillary acetic protein (GFAP) promoter has been
used to
selectively express genes in cells of glial origin.
[000177] Expression vectors used in eukaryotic host cells (yeast, fungi,
insect, plant,
animal, human or nucleated cells) may also contain sequences necessary for the
termination
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of transcription which may affect mRNA expression. These regions are
transcribed as
polyadenylated segments in the untranslated portion of the mRNA encoding
tissue factor
protein. The 3' untranslated regions also include transcription termination
sites. It is
preferred that the transcription unit also contain a polyadenylation region.
One benefit of
this region is that it increases the likelihood that the transcribed unit will
be processed and
transported like mRNA. The identification and use of polyadenylation signals
in
expression constructs is well established. It is preferred that homologous
polyadenylation
signals be used in the transgene constructs. In certain transcription units,
the
polyadenylation region is derived from the SV40 early polyadenylation signal
and consists
of about 400 bases. It is also preferred that the transcribed units contain
other standard
sequences alone or in combination with the above sequences improve expression
from, or
stability of, the construct.
(B) Markers
[000178] The viral vectors can include nucleic acid sequence encoding a marker
product.
This marker product is used to determine if the gene has been delivered to the
cell and once
delivered is being expressed. Preferred marker genes are the E. Coli lacZ
gene, which
encodes 13-galactosidase, and green fluorescent protein.
[000179] In some embodiments the marker may be a selectable marker. Examples
of
suitable selectable markers for mammalian cells are dihydrofolate reductase
(DHFR),
thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin.
When
such selectable markers are successfully transferred into a mammalian host
cell, the
transformed mammalian host cell can survive if placed under selective
pressure. There are
two widely used distinct categories of selective regimes. The first category
is based on a
cell's metabolism and the use of a mutant cell line which lacks the ability to
grow
independent of a supplemented media. Two examples are: CHO DHFR- cells and
mouse
LTK- cells. These cells lack the ability to grow without the addition of such
nutrients as
thymidine or hypoxanthine. Because these cells lack certain genes necessary
for a complete
nucleotide synthesis pathway, they cannot survive unless the missing
nucleotides are
provided in a supplemented media. An alternative to supplementing the media is
to
introduce an intact DHFR or TK gene into cells lacking the respective genes,
thus altering
their growth requirements. Individual cells which were not transformed with
the DHFR or
TK gene will not be capable of survival in non-supplemented media.
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[000180] The second category is dominant selection which refers to a selection
scheme
used in any cell type and does not require the use of a mutant cell line.
These schemes
typically use a drug to arrest growth of a host cell. Those cells which have a
novel gene
would express a protein conveying drug resistance and would survive the
selection.
Examples of such dominant selection use the drugs neomycin, (Southern P. and
Berg, P., J.
Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan, R.C. and
Berg, P.
Science 209: 1422 (1980)) or hygromycin, (Sugden, B. et al., Mol. Cell. Biol.
5: 410-413
(1985)). The three examples employ bacterial genes under eukaryotic control to
convey
resistance to the appropriate drug G418 or neomycin (geneticin), xgpt
(mycophenolic acid)
or hygromycin, respectively. Others include the neomycin analog G418 and
puramycin.
[000181] Other materials, including therapeutic, bioactive, diagnostic, and/or
prophylactic agents, cells or whole tissues, may be included in the long-
acting formulations
used in the present invention. These materials can be used, for example, for
controlled
release of a drug, to render the devices radio-opaque, stimulate tissue in-
growth, promote
tissue regeneration, prevent infection, or modify the porosity of the device.
[000182] Antibodies or nucleic acids may be complexed or otherwise associated
with
other excipients contained in the microparticle composition that alter or
enhance the
biological effect, biological activity, stability, or release of the antibody
or nucleic acid. In
another aspect of the present invention, these agents may simply be
incorporated into the
microparticle composition along with the antibody or nucleic acid without
otherwise
forming a complex or association between the antibody or nucleic acid and the
other agent.
Antibody or nucleic acids in the form of prodrugs (including polymeric
prodrugs) may be
incorporated into the microparticle compositions of the present invention.
Further aspects
of the present invention include the incorporation of antibody or nucleic
acids that have
been otherwise chemically modified (for example, for purposes of achieving
biological
targeting or for other means of affecting the pharmacokinetics or
biodistribution of the
native antibody or nucleic acid or any combinations of the above.)
[000183] Other components
[000184] Other components such as, for example, solvents, suspension agents,
surfactants, carriers, diluents, fillers, etc. that are typically used for the
delivery of a free
antibody or nucleic acid and for the delivery of long-acting formulations can
also be used
herein. One of skill in the art would know how to select the proper carrier to
deliver the
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free antibody or nucleic acid and the long-acting formulation. In various
aspects, the carrier
comprises water or is water.
[000185] In one specific aspect, the long-acting formulation can comprise an
antibody
dissolved in an aqueous solution and microencapsulated antibody suspended in
the same
aqueous solution. To effect release, the antibody is microencapsulated in 25-
to 125-micron
diameter microparticles containing a 85:15 poly(D,L-lactide-co-glycolide)
excipient with an
inherent viscosity of 0.7 dL/gm. The antibody content of the microparticles is
less than 5
wt% so the microencapsulated antibody is not released until the microparticle
excipient
begins to substantially hydrolyze and resorb. Upon administration of the
aqueous
composition comprising dissolved antibody and microencapsulated antibody, the
dissolved
antibody first affords efficacy for 3 months. During this 3-month period, the
microencapsulated antibody is not released and stays within the
microparticles. However,
after 3 months, the 85:15 poly(D,L-lactide-co-glycolide) excipient begins to
substantially
hydrolyze and resorb allowing for release of the microencapsulated antibody
for the next 3
months.
[000186] Although several aspects of the present invention have been described
in the
detailed description, it should be understood that the invention is not
limited to the aspects
disclosed, but is capable of numerous rearrangements, modifications and
substitutions
without departing from the spirit of the invention as set forth and defmed by
the following
claims.
[000187] EXAMPLES
[000188] To further illustrate the principles of the present invention, the
following
examples are put forth so as to provide those of ordinary skill in the art
with a complete
disclosure and description of how the compositions, articles, devices, and
methods claimed
herein are made and evaluated. They are intended to be purely exemplary of the
invention
and are not intended to limit the scope of what the inventors regard as their
invention.
Efforts have been made to ensure accuracy with respect to numbers (e.g.,
amounts,
temperatures, etc.); however, some errors and deviations should be accounted
for. Unless
indicated otherwise, temperature is C or is at ambient temperature, and
pressure is at or
near atmospheric. There are numerous variations and combinations of process
conditions
that can be used to optimize product quality and performance. Only reasonable
and routine
experimentation will be required to optimize such process conditions.
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[000189] 1. PLG microparticle formulation of an antibody (prophetic example).
[000190] An antibody is dispersed using a Polytron mixer into 2-mL of a
solution of 10%
85:15 DL-PLG (inherent viscosity 0.4 dL/g) in methylene chloride at a loading
level of 5%
by weight of the antibody relative to the combined weight of drug and polymer.
Mixing is
performed at 2-4 c for 15-20 seconds at which time the suspension is
transferred into a 5-cc
syringe. Using an 18-gauge needle, this suspension is then delivered to an in-
line Silverson
mixer at a rate of about 10 g/min at the same time that an aqueous solution
consisting of 2
wt % poly(vinyl alcohol) (PVA) saturated with methylene chloride solvent. The
PVA
solution is delivered to the in-line mixer at a flow rate of about 50 g/min.
The resulting
emulsion is then immediately diluted with fresh distilled water delivered at a
flow rate of
about 250 g/min in an extraction coil placed downstream from the Silverson
mixer. The
effluent from the extraction coil is then transferred to a tank that is
stirred at about 600 rpm.
The total volume of effluent from the process is stirred for 2-4 hours to
facilitate extraction
of solvent from the suspension and to, thereby, harden the antibody-loaded
polymer
microparticles. After hardening, the microparticles are isolated by passing
the effluent
through 125-micron and 25-micron sieves. The product collected on the 25-
micron is then
washed with an excess volume of fresh, distilled water (for example, 4-6 L).
The product is
then dried at ambient pressure and temperature by placing the 25-micron sieve
under a
laminar flow hood for at least 12 hours. The product is then gently scraped
off of the sieve
and is stored desiccated and frozen.
[000191] For ocular administration, the antibody-loaded microparticles are
combined
with a 50 to 100-microliter solution of antibody. The mixture is then injected
into the
vitreous of the eye. Upon administration, the unencapsulated antibody has
efficacy for 1
month or longer. During this time, the PLG microparticles release little or no
antibody.
After the unencapsulated antibody is gone or no longer efficacious, the
microencapsulated
antibody begins to release. This release of microencapsulated antibody can be
designed to
occur for days, weeks of months. The release of microencapsulated antibody is
therefore
delayed until the unencapsulated antibody is no longer efficacious. The use of
unencapsulated antibody means less microencapsulation polymer is needed and
therefore
more bioactive can be administered in the designated 50 to 100-uL volume.
[000192] Various modifications and variations can be made to the compositions,
articles,
devices, and methods described herein. Other aspects of the compositions,
articles, devices,
and methods described herein will be apparent from consideration of the
specification and
SUBSTITUTE SHEET (RULE 26)
CA 02690858 2009-12-07
WO 2008/153997 PCT/US2008/007216
practice of the compositions, articles, devices, and/or methods disclosed
herein. It is
intended that the specification and examples be considered as exemplary.
56
SUBSTITUTE SHEET (RULE 26)
